Compositions and methods for regulation of steroidogenesis

ABSTRACT

Compositions and methods relating to the regulation of transport of cholesterol into the mitochondria of a cell and, therefore, for the regulation of steroidogenesis are provided. Compositions include nucleic acid molecules encoding a steroidogenic acute regulatory protein (StAR), StAR protein molecules and peptides having amino acid sequences as disclosed herein, and anti-StAR antibodies. Methods include immunoassays using anti-StAR antibodies and nucleic acid based screening methods for pathologies correlated with defects in StAR, such as lipoid congenital adrenal hyperplasia and dose sensitive sex reversal. In addition, these compositions and methods may be useful for treatment of steroid honnone-dependent disorders, in particular, for lipoid congenital adrenal hyperplasia.

The present application is a continuation-in-part application of U.S.Ser. No. 08/530,960 filed Nov. 4, 1995, now U.S. Pat. No. 5,872,230.That disclosure is incorporated herein by ence in its entirety. Thegovernment owns certain rights in the present invention pursuant togrant numbers HD 17481 and HD07688 from the National Institutes ofHealth.

FIELD OF THE INVENTION

The present invention relates generally to compositions and methods forthe regulation of steroidogenesis. More particularly, it concernscompositions and methods relating to the regulation of transport ofcholesterol into the mitochondria of a cell for the synthesis ofandrogens, estrogens, glucocorticoids, mineralocorticoids, andprogestagens. The invention also relates to methods for detecting andtreating steroid hormone-dependent disorders. The nucleic acid moleculesof the present invention also provide methods for screening a sample forsteroid hormone-dependent disorders, as well as to methods for preparingrecombinant proteins for StAR. The invention further relates to thefield of nucleic acid probes and primers, as the various nucleic acidmolecules of the invention may be used as molecular probes in all of theaforedescribed methods, as well as primers for amplifying particularsequences of interest.

BACKGROUND OF THE INVENTION

The testis is known to be the source of circulating androgens that areresponsible for the maintenance of the secondary sexual characteristicsin the male. In most species the testis has two separate compartments:the seminiferous tubules that contain the Sertoli cells, the peritubularcells, and the germ cells; and the interstitial compartment thatcontains the Leydig cells, macrophages, lymphocytes, granulocytes andthe cells composing the blood, nerve and lymphatic structures.

The Leydig cells, located in the interstitial compartment and comprisingapproximately 2-3% of the total testicular cell number in most species,are the only cells in the testis that contain two key steroidogenicenzymes, namely, cytochrome P450 side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3 beta-HSD). Thus, Leydig cells arethe only testicular cells capable of the first two steps insteroidogenesis; i) the conversion of cholesterol, the substrate for allsteroid hormones, to pregnenolone; and ii) conversion of pregnenolone toprogesterone. Therefore, the interstitial compartment in general, andthe Leydig cells in particular synthesize virtually all of the steroidsproduced in the testis, with testosterone being the major steroidbiosynthesized.

The major stimulus for the biosynthesis of testosterone in the Leydigcell is the gonadotrophic hormone, luteinizing hormone (LH). LH issecreted from specific cells located in the anterior pituitary and itinteracts with specific receptors on the surface of the Leydig cell andinitiates the signal for testosterone production. Cellular events occurrapidly in response to the trophic hormone stimulation of Leydig cells,and result in the synthesis and secretion of testosterone. These rapidor acute effects of hormone stimulation occur within minutes and can bedistinguished temporally from the slower chronic effects that occur onthe order of many hours and that involve mechanisms to increase genetranscription and translation of the steroid hydroxylase cytochrome P450enzymes involved in the biosynthesis of these steroids.

The rate-limiting enzymatic step in steroidogenesis is the conversion ofcholesterol to pregnenolone by the cholesterol side-chain cleavagecomplex (CSCC) which is localized to the mitochondrial inner membrane(Stone and Hechter, 1954; Karaboyas and Koritz, 1965; Simpson, et al.1972). However, the delivery of the substrate cholesterol from cellularstores and the outer mitochondrial membrane to the inner mitochondrialmembrane and the CSCC is the true regulated, rate-limiting step in thisprocess (Crivello and Jefcoate, 1980; Jefcoate, et al., 1987).Cycloheximide, an inhibitor of protein synthesis, blocks thehormone-induced steroid production in two steroidogenic tissues of therat; the adrenal and testis (Ferguson, 1963; Garren, et al., 1965; Davisand Garren, 1968; Jefcoate et al., 1974; Mendelson et al., 1975; Cooke,et al., 1975). This block is at the point of transfer of cholesterolfrom the outer to the inner mitochondrial membrane and the CSCC (Simpsonet al., 1972; Privalle et al. 1983). Therefore, acute regulation ofsteroidogenesis requires de novo protein synthesis (Jefcoate et al.,1986).

During protein import into the mitochondrial matrix, the inner and outermitochondrial membranes become closely associated and form proteintranslocation “contact sites” (Schleyer and Neupert, 1985; Schwaiger etal., 1987; Glick, et al., 1991). Phospholipids are transferred from theouter mitochondrial membrane to the inner mitochondrial membrane atthese membrane “contact sites” (Simbeni et al., 1990; Simbeni et al.,1991; Ardail et al., 1991). Therefore, the intramitochondrialcholesterol translocation required for steroidogenesis may also occur atmembrane contact sites. An increase in intrrmitochondrial membranecontacts by a hormone-dependent, cycloheximide-sensitive mechanism mayregulate cholesterol transport to the CSCC (Jefcoate, et al., 1986).Thus, in the acute regulation of steroidogenesis, a putative functionfor the newly synthesized regulatory protein may be to facilitate theformation of mitochondrial contact sites that would result in anincreased rate of transfer of cholesterol to the inner membrane and CSCCwhich ultimately would result in the observed increase in the rate ofsteroid production. However, the seareh for thesecycloheximide-sensitive regulatory protein(s) has been ongoing fornearly 30 years, but, as yet, the mechanism of cholesterol transfer tothe CSCC is not known.

The present inventors have previously identified a family ofhormone-induced mitochondrial proteins in MA-10 cells that regulatecholesterol delivery to the inner mitochondrial membrane and the CSCC.These proteins have been described as the mitochondrial 37 kDa, 32 kDa,and 30 kDa molecular weight proteins and they are synthesized inresponse to either LH and hCG or by stimulation with the CAMP analogue,Bt2cAMP (Stocco and Kilgore, 1988). The 30 kDa species consists of fourseparate proteins and proteolytic digestion of all four forms indicatesthat they are all modified forms of the same protein (Stocco and Chen,1991). Pulse chase experiments and tryptic peptide mapping of the 37 kDaand 3OkDa proteins indicated that the 37 kDa form is a precursor to the30 kDa protein (Stocco and Sodeman, 1991; Epstein and Orne-Johnson,1991). These reports, however, lack information regarding the structureof the nucleic acid molecules and protein molecules involved insteroidogenesis.

Lipoid congenital adrenal hyperplasia (LCAH) is a lethal autosomalrecessive disease that results in a complete inability of a newborninfant to synthesize steroids. The lack of mineralocorticoids andglucocorticoids results in death within days to weeks of birth if notdetected and treated with adequate steroid hormone replacement therapy.This condition is manifested by the presence of large adrenalscontaining very high levels of cholesterol and cholesterol esters andalso by an increased amount of lipid accumulation in testicular Leydigcells, though this level is somewhat lower than that seen in adrenals.As isolated, mitochondria from adrenals and gonads of affected patientscannot convert cholesterol to pregnenolone (Camacho et al., 1968;Degenhart et al., 1972; Koizumi et al., Hauffa et al., 1985). TheP450scc enzyme that converts cholesterol to pregnenolone has been shownto be nornal in patients who suffered from this disease (Lin et al.,1991). Thus, the defect lies upstream of P450scc at the point ofcholesterol delivery to the enzyme.

Prior art lacks sufficient identification of the agent(s) responsiblefor the LCAH metabolic defect and defects in cholesterol transport,lacks screening methods for their detection, and lacks provision ofpharmacological agents effective in alleviating the defects. Because ofthese problems, known procedures are not completely satisfactory despiteefforts of persons skilled in the art, and the present inventors havesearched for improvements. Further characterization of agents involvedin these defects at the amino acid and nucleic acid levels would providepotential solutions and alternatives to resolving these and otherproblems in the art.

SUMMARY OF THE INVENTION

The present invention seeks to overcome these and other drawbacksinherent in the prior art by providing compositions and methodsincluding the nucleotide sequence of the gene encoding a steroidogenicacute regulatory protein (StAR) protein, or compositions that include aprotein having the amino acid sequence of the StAR protein. Thefindamental importance of the StAR protein is that it is the acuteregulator of a key step in the steroidogenic biosynthetic pathway.Importantly, the production of mineralocorticoids, glucocorticoids, andsex hormones are dependent on the expression of this protein.

Nucleic acid molecules having nucleotide sequences of the gene encodingStAR may be used in a variety of different diagnostic applications,including the evaluation of gene defects associated with steroid hormoneproduction. The hormonally induced, CAMP-mediated acute regulation ofsteroid hormone biosynthesis in steroidogenic cells is characterized bythe mobilization of cholesterol from cellular stores to the mitochondriaouter membrane, and its translocation to the inner membrane where theconversion of cholesterol to pregnenolone occurs. Steroidhormone-dependent disorders that may be addressed using compositions andmethods of the present invention include lipoid congenital adrenalhyperplasia, infertility, sexual maturation, androgen-responsive tumors,precocious puberty, McCune-Albright syndrome, adrenal-hypoplasiacongenita, or hypogonadotropic hypogonadism.

Further, in pregnancy induced diabetes, progesterone levels are lowerthan normal and the fetus may be aborted spontaneously. The level ofStAR protein may be deficient in these patients and it may be possibleas a result of the present invention to monitor levels of StAR inpregnancy for predicting patients that may be at risk for spontaneousabortion.

The nucleic acid molecules of the invention may further be used toprovide recombinant preparations of the StAR protein. These highlypurified preparations can also be provided in relatively high yield inconjunction with techniques well known to those of skill in the art ofrecombinant technology.

In certain aspects, the invention relates to a purified nucleic acidmolecule having a nucleotide sequence encoding a steroidogenic acuteregulatory protein, the protein having an amino acid sequence as setforth in SEQ ID NO: 2 or SEQ ID NO: 18, or sequences that are about 80%to about 99% identical to that sequence. “Purified” nucleic acidmolecules having a nucleotide sequence encoding steroidogenic acuteregulatory protein (StAR), in some embodiments of the invention, means aStAR encoding nucleic acid molecule substantially free of nucleic acidmolecules not encoding an amino acid sequence having about 80% to about99% identity to the sequence set forth in SEQ ID NO:2 or SEQ ID NO: 18.A further embodiment of the invention is a purified nucleic acidmolecule having a nucleotide sequence encoding a steroidogenic acuteregulatory protein antigen, the antigen having an amino acid sequencehaving about 80% to about 99% identity to the sequence set forth in SEQID NO:8, and the nucleic acid molecule being substantially free ofnucleic acid molecules not encoding the steroidogenic acute regulatoryprotein antigen.

The term “essentially as set forth in SEQ ID NO:2, SEQ ID NO:18 or SEQID NO:8” means that the sequence substantially corresponds to a portionof the indicated sequence and has relatively few amino acids which arenot identical to, or a biologically flimctional equivalent of, the aminoacids set forth. The term “biologically functional equivalent” is wellunderstood in the art and is further defined as a protein having asequence essentially as set forth in the indicated SEQ ID NO, and thatis involved in the transfer of cholesterol from cellular stores to theinner mitochondrial membrane. Accordingly, sequences which have betweenabout 70% and about 80%; or more preferably, between about 81% and about90%; or even more preferably, between about 91% and about 99%; of aminoacids that are identical or functionally equivalent to the amino acidsof the indicated SEQ ID NO:2, 18, or 8 will be sequences which are“essentially as set forth in SEQ ID NO:2, 18, or 8”.

Some embodiments of the above-described nucleic acid molecule are thosewherein the steroidogenic acute regulatory protein has the amino acidsequence of SEQ ID NO: 2 or 18. In ftrther defined embodiments, theinvention provides the above-described nucleic acid molecule wherein theamino acid sequence begins with amino acid methionine at position 48 ofSEQ ID NO:2 and extends through amino acid cysteine at position 284 ofSEQ ID NO:2. Amino acids 1-47 constitute the signal sequence which iscleaved during processing to the mature protein as described in Example2.

A further embodiment of the present invention is where the nucleic acidmolecule has a nucleotide sequence as set forth in SEQ ID NO: 1 or 19,and preferably, the nucleic acid molecule is further defined asincluding a detectable label.

Nucleic Acids

Some embodiments of the present invention provide purified nucleic acidmolecules that encode StAR protein having an amino acid sequenceessentially as set forth in SEQ ID NO:2 or 18. As used herein, the terms“nucleic acid molecule” may refer to a DNA or RNA molecule which hasbeen isolated free of total genomic DNA, or free of total RNA, of aparticular species. Therefore, a “purified” nucleic acid molecule asused herein, refers to a nucleic acid molecule that contains a StARcoding sequence yet is isolated away from, or purified free from, totalgenomic DNA or total RNA, for example, total human genomic DNA. Includedwithin the term “DNA molecule”, are DNA segments and smaller fragmentsof such segments, and also recombinant vectors, including, for example,plasmids, cosmids, phage, viruses, and the like.

Another embodiment of the present invention is a purified nucleic acidmolecule, further defined as including a nucleotide sequence inaccordance with SEQ ID NO: 1 or 19. In some embodiments, the purifiednucleic acid segment comprises a nucleotide sequence having about 75% toabout 99% identity with the sequence at SEQ ID NO: 1 or with the StARcoding sequences thereof (see FIG. 3). Such nucleotide sequences aremore particularly defined as being substantially free of nucleic acidsnot encoding the StAR protein.

Similarly, a DNA molecule comprising an isolated or purified StAR generefers to a DNA molecule including StAR coding sequences isolatedsubstantially away from other naturally occurring genes or proteinencoding sequences. In this respect, the term “gene” is used forsimplicity to refer to a functional protein, polypeptide or peptideencoding unit. As will be understood by those in the art, thisfunctional term includes both genomic sequences, cDNA sequences orcombinations thereof. “Isolated substantially away from other codingsequences” means that the gene of interest, in this case the StARencoding gene, forms the significant part of the coding region of theDNA molecule, and that the DNA molecule does not contain large portionsof naturally-occurring coding DNA, such as large chromosomal fragmentsor other finctional genes or cDNA coding regions. Of course, this refersto the DNA molecule as originally isolated, and does not exclude genesor coding regions later added to the segment by the hand of man.

Another embodiment of the present invention is a purified nucleic acidmolecule that encodes a protein having a sequence with about 80% toabout 99% identity to SEQ ID NO:2 or 18, further defined as arecombinant vector. As used herein, the term “recombinant vector”,refers to a vector that has been modified to contain a nucleic acidsegment that encodes a StAR protein, or fragment of interest thereof Therecombinant vector may be further defined as an expression vectorcomprising a promoter operatively linked to said StAR encoding nucleicacid molecule. In some embodiments, the recombinant vector comprises anucleic acid sequence in accordance with SEQ ID NO:1 or 19, or withabout 80% to about 99% identity to the sequence of SEQ ID NO:1 or 19. Byway of example and not limitation, vectors may be further defined as apCMV, pUC and derivatives thereof, SV40, adenoviral, retroviral, yeastplasmids, Baculovirus or Vaccinia virus vector.

A further preferred embodiment of the present invention is a host cell,made recombinant with a recombinant vector comprising a StAR gene. Therecombinant host cell may be a prokaryotic or a eukaryotic cell. In amore preferred embodiment, the recombinant host cell is a eukaryoticcell. As used herein, the term “engineered” or “recombinant” cell isintended to refer to a cell into which a recombinant gene, such as agene encoding StAR, has been introduced. Therefore, engineered cells aredistinguishable from naturally occurring cells which do not contain arecombinantly introduced gene. Thus, engineered cells are cells having agene or genes introduced through the hand of man. Recombinantlyintroduced genes will either be in the form of a cDNA gene (i.e., theywill not contain introns), a copy of a genomic gene, or will includegenes positioned adjacent to a promoter not naturally associated withthe particular introduced gene, or combinations thereof. Preferred hostcells may be further defined as a Leydig cell (primary or MA-10cells),an adrenalcortical cell such as the H295 human adrenalcortical cellline, a primary culture ovarian granulosa cell, a COS cell,Saccharomyces cerevisiae, or Escherichia coli cell.

Generally speaking, it may be more convenient to employ as therecombinant gene a cDNA version of the gene. It is believed that the useof a cDNA version will provide advantages in that the size of the genewill generally be much smaller and more readily employed to transfectthe targeted cell than will a genomic gene, which will typically be upto an order of magnitude larger than the cDNA gene. However, theinventors do not exclude the possibility of employing a genomic versionof a particular gene where desired.

In certain embodiments, the invention concerns isolated DNA moleculesand recombinant vectors which encode a protein or peptide that includeswithin its amino acid sequence an amino acid sequence essentially as setforth in SEQ ID NO:2 or 18. Naturally, where the DNA segment or vectorencodes a fall length StAR protein, or is intended for use in expressingthe StAR protein, the most preferred sequences are those which areessentially as set forth in SEQ ID NO:2 or 18.

In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence anucleic acid sequence essentially as set forth in SEQ ID NO: 1 or 19.The term “essentially as set forth in SEQ ID NO: 1 or 19”, is used inthe same sense as described above and means that the nucleic acidsequence substantially corresponds to a portion of SEQ ID NO: 1 or 19,and has relatively few codons which are not identical, or functionallyequivalent, to the codons of SEQ ID NO: 1 or 19. The term “functionallyequivalent codon” is used herein to refer to codons that encode the sameamino acid, such as the six codons for arginine or serine, as set forthin Table 1, and also refers to codons that encode biologicallyequivalent amino acids.

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences which may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes. The nucleic acid segments of the present invention,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably.

Excepting intronic or flanking regions, and allowing for the degeneracyof the genetic code, sequences which have between about 70% and about90%; or more preferably, between about 80% and about 90%; or betweenabout 80% and about 99%; of nucleotides which are identical to thenucleotides of SEQ ID NO:1 or 19 will be sequences which are“essentially as set forth in SEQ ID NO:1 or 19”. Sequences which areessentially the same as those set forth in SEQ ID NO: 1 or 19 may alsobe functionally defined as sequences which are capable of hybridizing toa nucleic acid segment containing the complement of SEQ ID NO: 1 or 19under relatively stringent conditions. Suitable relatively stringenthybridization conditions will be well known to those of skill in the artand are clearly set forth herein, for example conditions for use withNorthern blot analysis, and as described in the various embodiments ofthe invention provided herein and particularly in Example 2.

TABLE 1 CODON DEGENERACY Amino Acids Codons Alanine Ala A GCA GCC GCGGCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid GluE GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

The present invention includes a purified nucleic acid moleculecomplementary, or essentially complementary, to the nucleic acidmolecule having the sequence set forth in SEQ ID NO: 1 or 19. Nucleicacid sequences which are “complementary” are those which are capable ofbase-pairing according to the standard Watson-Crick complementarityrules. As used herein, the term “complementary sequences” means nucleicacid sequences which are substantially complementary, as may be assessedby the same nucleotide comparison set forth above, or as defined asbeing capable of hybridizing to the nucleic acid segment of SEQ ID NO:lor 19 under relatively stringent conditions such as those describedherein in the detailed description of the preferred embodiments.Complementary nucleotide sequences are useful for detection andpurification of hybridizing nucleic acid molecules. A preferredembodiment of the invention is a molecule complementary to SEQ ID NO: 1or 19 and is a cDNA molecule complementary to a steroidogenic acuteregulatory protein MnRNA.

The present inventors also envision the preparation of fusion proteinsand peptides, e.g., where the StAR coding regions are aligned within thesame expression unit with other proteins or peptides having desiredfunctions, such as for purification or immunodetection purposes (e.g.,proteins which may be purified by affinity chromatography and enzymelabel coding regions, respectively).

StAR protein has been successfully expressed in eukaryotic expressionsystems by the present inventors, for example, using the PCMV vector inCOS cells. Other expression systems contemplated by the presentinventors include, e.g., baculovirus-based, glutamine synthase-based,dihydrofolate reductase-based systems, or the like. For expression inthis manner, one would position the coding sequences adjacent to andunder the control of the promoter. It is understood in the art that tobring a coding sequence under the control of such a promoter, onepositions the 5′ end of the transcription initiation site of thetranscriptional reading frame of the protein between about 1 and about50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter.

Where eukaryotic expression is contemplated, one will also typicallydesire to incorporate into the transcriptional unit which includes theStAR gene, an appropriate polyadenylation site (e.g., 5′-AATAAA-3′) ifone was not contained within the original cloned segment. Typically, thepoly A addition site is placed about 30 to 2000 nucleotides “downstream”of the termination site of the protein at a position prior totranscription termination.

It is contemplated that virtually any of the commonly employed hostcells can be used in connection with the expression of StAR inaccordance herewith. Examples include cell lines typically employed foreukaryotic expression such as COS, CHO, MA-10 cells, or Saccharomycescerevisiae.

It is proposed that transformation of host cells with DNA segmentsencoding the StAR protein will provide a convenient means for obtainingpurified StAR protein. It is also proposed that cDNA, genomic sequences,and combinations thereof, are suitable for eukaryotic expression, as thehost cell will process the genomic transcripts to yield functional mRNAfor translation into protein. Other embodiments of the inventioncomprise compositions comprising a purified RNA molecule correspondingto StAR. Such embodiments, by way of example, have a nucleotide sequenceof SEQ ID NO:14, 15, 16, or 17.

Nucleic Acid Hybridization and PCR reactions.

Oligonucleotide sequences based on the mouse or a homologous sequence ofStAR may be used as primers in a polymerase chain reaction to screen forpossible mutations in StAR MRNA causing a variety of pathologies, forexample, the lethal human disease, lipoid congenital adrenalhyperplasia. Therefore, StAR nucleic acid sequence can be applied toscreen prenatal, perinatal, or neonatal DNA for possible mutations inStAR. If the disease is detected early, then continualmineralocorticoid, glucocorticoid, or steroid replacement therapy canprolong the life of the patient. Further applications will arise whenadditional disease states are linked to mutations in the StAR gene, orunder conditions where mutations in related genes result in decreasedlevels of StAR MRNA or protein. In these cases, analysis of StAR MRNA orprotein has significant diagnostic value.

DNA probes and primers useful in hybridization studies and PCR reactionsmay be derived from any portion of SEQ ID NO: 1 or 19 and are generallyat least about seventeen nucleotides in length. Therefore, probes andprimers are specifically contemplated that comprise nucleotides 1 to 17,or 2 to 18, or 3 to 19 and so forth up to a probe comprising the last 17nucleotides of the nucleotide sequence of SEQ ID NO: I or 19. Thus, eachprobe would comprise at least about 17 linear nucleotides of thenucleotide sequence of SEQ ID NO:1 or 19, designated by the formula “nto n+16,” where n is an integer from 1 to about 1435. Longer probes thathybridize to the StAR gene under low, medium, medium-high and highstringency conditions are also contemplated, including those thatcomprise the entire nucleotide sequence of SEQ ID NO: 1 or 19. Selectedoligonucleotide subportions of the gene encoding StAR have significantutility irrespective of whether they encode antigenic peptides. In theseaspects, it is contemplated, for example, that shorter or longer nucleicacid fragments of the StAR gene, prepared synthetically, recombinantly,or otherwise, can be employed as hybridization probes. Such probes canbe readily employed in a variety of ways, including their use in theidentification of genes encoding StAR in biological tissues or clinicalsamples, as well as in the detection and evaluation of StAR inpathologies that relate to cholesterol and/or steroid synthesis.Biological or clinical samples include, but are not limited to, biopsyspecimens from adrenal or gonadal tissue, or blood, for example.

A general method for preparing oligonucleotides of various lengths andsequences is described by Caracciolo et al. (1989). In general, thereare two commonly used solid phase-based approaches to the synthesis ofoligonucleotides containing conventional 5′ -3′ linkages, one involvingintermediate phosphoramidites and the other involving intermediatephosphonate linkages. In the phosphoramidite synthesis a suitablyprotected nucleotide having a cyanoethylphosphoramidate at the positionto be coupled is reacted with the free hydroxyl of a growing nucleotidechain derivatized to a solid-support. The reaction yields acyanoethylphosphite, which linkage must be oxidized to thecyanoethylphosphate at each intermediate step, since the reduced form isunstable to acid.

The phosphonate based synthesis is conducted by the reaction of asuitably protected nucleotide containing a phosphonate moiety at aposition to be coupled with a solid phase-derivatized nucleotide chainhaving a free hydroxyl group, in the presence of a suitable activator toobtain a phosphonate ester linkage, which is stable to acid. Thus, theoxidation to the phosphate or thiophosphate can be conducted at anypoint duing synthesis of the oligonucleotide or after synthesis of theoligonucleotide is complete.

The phosphonates can also be converted to phosphoramidate derivatives byreaction with a primary or secondary amine in the presence of carbontetrachloride. To indicate the two approaches generically, the incomingnucleotide is regarded as having an “activated” phosphite/phosphategroup. In addition to employing commonly used solid phase synthesistechniques, oligonucleotides may also be synthesized using solutionphase methods such as triester synthesis. The methods are workable, butin general, less efficient for oligonucleotides of any substantiallength.

Preferred oligonucleotides resistant to in vivo hydrolysis may contain aphosphorothioate substitution at each base. Oligodeoxynucleotides ortheir phosphorothioate analogues may be synthesized using an AppliedBiosystem 380B DNA synthesizer (Applied Biosystems, Inc., Foster City,Calif.).

A furlher embodiment of the invention is a purified nucleic acidmolecule having at least a 17, 20, 25, 30, 50, 100, 200, 500, or 1000nucleotide sequence that corresponds to, or is capable of hybridizing tothe nucleic acid sequence of SEQ ID NO:1 or 19 under conditions standardfor hybridization fidelity and stability. Furthermore, it iscontemplated that nucleic acid molecules having a nucleotide sequence ofSEQ ID NOS:1, 9, 10, 11, 12, 13, or 19 for stretches of between about 10nucleotides to about 20 or to about 30 nucleotides will find particularutility, with even longer sequences, e.g., 40, 50, 150, 250, 450, evenup to fill length, being more preferred for certain embodiments. Theability of such nucleic acid probes to specifically hybridize to StARnucleic acid sequences will enable them to be of use in a variety ofembodiments. For example, the probes can be used in a variety of assaysfor detecting the presence of complementary sequences in a given sample.However, other uses are envisioned, including the use of the sequenceinformation for the preparation of mutant species primers, or primersfor use in preparing other genetic constructions.

These probes will be useful in hybridization embodiments, such asSouthern and Northern blotting. The total size of fragment, as well asthe size of the complementary stretch(es), will ultimately depend on theintended use or application of the particular nucleic acid segment.Smaller fragments will generally find use in hybridiaation embodiments,wherein the length of the complementary region may be varied, such asbetween about 20 and about 40 nucleotides, or even up to the full lengthof the nucleic acid as shown in SEQ ID NOS: 1, 9-13, or 19 according tothe complementary sequences one wishes to detect.

The use of a hybridization probe of about 10 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having complementary sequences over stretchesgreater than 10 bases in length are preferred, though, in order toincrease stability and selectivity of the hybrid, and thereby improvethe quality and degree of specific hybrid molecules obtained. One willgenerally prefer to design nucleic acid molecules havinggene-complementary stretches of 15 to 20 nucleotides, or even longerwhere desired. Such fragments may be readily prepared by, for example,directly synthesizing the fragment by chemical means, by application ofnucleic acid reproduction technology, such as the PCR technology of U.S.Pat. No. 4,683,202 (herein incorporated by reference) or by introducingselected sequences into recombinant vectors for recombinant production.

Depending on the application envisioned, one will desire to employvarying conditions of hybridization to achieve varying degrees ofselectivity of probe towards target sequence. For applications requiringhigh selectivity, one will typically desire to employ relativelystringent conditions to form the hybrids, e.g., one will selectrelatively low salt andor high temperature conditions, such as providedby 0.02M-0.15M NaCl at temperatures of 50° C. to 70° C. Such selectiveconditions tolerate little, if any, mismatch between the probe and thetemplate or target strand.

Where one desires to prepare mutants employing a mutant primer strandhybridized to an underlying template or where one seeks to isolatesequences from related species, functional equivalents, or the like,less stringent hybridization conditions will typically be needed inorder to allow formation of the heteroduplex. In these circumstances,one may desire to employ conditions such as 0.15M-0.9M salt, attemperatures ranging from 20° C. to 55° C. Cross-hybridizing species canthereby be readily identified as positively hybridizing signals withrespect to control hybridizations. In any case, it is generallyappreciated that conditions can be rendered more stringent by theaddition of increasing amounts of formamide, which serves to destabilizethe hybrid duplex in the same manner as increased temperature. Thus,hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmental undesirable reagents. In the case of enzyme tags,colorimetric indicator substrates are known which can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C contents, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label.

Longer DNA segments will often find particular utility in therecombinant production of peptides or proteins. DNA segments whichencode peptides from about 15 to about 50 amino acids in length, or morepreferably, from about 15 to about 30 amino acids in length comprisingthe amino acid sequence of SEQ ID NO:8 are contemplated to beparticularly useful. DNA segments encoding peptides will generally havea minimum coding length in the order of about 45 to about 150, or toabout 90 nucleotides. DNA segments encoding full length proteins mayhave a minimum coding length in the order of about 2000 nucleotides fora protein or otherwise biologically active equivalent peptide having atleast a sufficient portion of the sequence in accordance with SEQ IDNO:2 or 18 capable of providing said StAR-biological activity.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis contemplated that a nucleic acid fragment of almost any length may beemployed, with the total length preferably being limited by the ease ofpreparation and use in the intended recombinant DNA protocol. Forexample, nucleic acid fragments may be prepared in accordance with thepresent invention which are up to 10,000 base pairs in length, withsegments of 5,000 or 3,000 being preferred and segments of about 1,000base pairs in length being particularly preferred. It will be understoodthat this invention is not limited to the particular nucleic acid andamino acid sequences having sequence identifiers as listed in Table 2.Therefore, DNA segments prepared in accordance with the presentinvention may also encode biologically functional equivalent proteins orpeptides which have variant amino acid sequences. Such sequences mayarise as a consequence of codon redundancy and fimctional equivalencywhich are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor peptides may be constructed via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged.

Any of a variety of steroidogenic cells may be used as a source toprepare the purified StAR protein of the invention having an amino acidsequence essentially as set forth in SEQ ID NO:2 or sequences havingsubstantial identity thereto. Substantial identity as used in thedefinition of the present invention is intended to mean amino acidsequences or nucleic acid sequences that have an about 75% to about 99%identical sequence to that of the referenced SEQ ID NO. For example,such a relationship exists between SEQ ID NO:2 (amino acid—mouse) and(amino acid—human) and SEQ ID NO:l (nucleic acid—mouse) and SEQ ID NO:19(nucleic acid —human). By way of example, particularly useful cellsinclude adrenal fasciculata, adrenal glomerulosa, corpus luteum cells,ovarian theca, ovarian granulosa, mouse Y-1 adrenalcortical tumor cells,primary Leydig cell cultures and MA-10 Leydig tumor cells. The cell lineemployed to prepare a mitochondrial extract for purposes of isolatingthe herein described StAR protein may comprise Leydig cell cultures andMA-10 Leydig tumor cells.

Table 2 lists the identity of sequences of the present disclosure havingsequence identifiers.

TABLE 2 Identification of Sequences Having Sequence Identifiers SEQ IDNO: IDENTITY 1 DNA sequence encoding StAR (mouse) 2 Protein sequence of30kDa StAR (mouse) 3 peptide 23 (mouse) 4 peptide 25 (mouse) 5 peptide45 (mouse) 6 Asn Gln Glu Gly Trp Lys 7 Ala Glu His Gly Pro Thr Cys MetVal 8 amino acids 88-98 of SEQ ID NO:2 (mouse) 9 degenerateoligonucleotides made to peptide 23; coding direction (mouse) 10degenerate oligonucleotides made to peptide 23; noncoding direction(mouse) 11 degenerate oligonucleotides made to peptide 25; codingdirection (mouse) 12 degenerate oligonucleotides made to peptide 25;noncoding direction (mouse) 13 PCR product, nucleotides 343-743 of SEQID NO:1 (mouse) 14 RNA sequence encoding StAR (mouse) 15 RNA sequenceencoding StAR peptide (human), (nucleic acid position 267-988 codingregion) 16 RNA sequence encoding StAR peptide (human) (nucleic acidposition 127-260 coding region). 17 RNA sequence for human noncodingStAR region (nucleic acid position 1051-1069). 18 Amino acid sequenceencoding StAR (human) (amino acid 1-285) 19 DNA sequence encoding StAR(human)

StAR Protein Compositions

In particular aspects, the present invention provides a purified StARprotein having an amino acid sequence essentially as set forth in SEQ IDNO:2 or 18. In a further embodiment of the composition, the amino acidsequence begins at the amino acid methionine at position 48 of SEQ IDNO: 2 and extends through amino acid cysteine at position 284 of SEQ IDNO:2.

The StAR protein may be phosphorylated or unphosphorylated. The mature30 kDa form of StAR protein has four different isoelectric species,designated as 30 kDa 1, 2, 3, and 4, with 1 being the most basic formand 4 the most acidic form. Studies by the present inventorsdemonstrated that forms 3 and 4 were phosphorylated forms of 1 and 2,and that phosphorylation is important for biological activity. Theseforms of the 30 kDa protein are usefull as molecular weight standards,and as standards for isoelectric focusing. Threonine, serine, andtyrosine amino acids are most frequently those amino acids in a proteinthat are phosphorylated, and in the case of the StAR protein, athreonine may be phosphorylated.

The purified 37 kDa StAR protein is expected to have many differentuses, including, for example, supplementing a patient lacking StARactivity to provide proper cholesterol transport and subsequentsynthesis of steroids.

In some aspects of the peptides of the StAR protein, the peptidescomprise an amino acid sequence in accordance with SEQ ID NO:3, 4, 5, 6,7, or 8. These peptides are useful for designing oligonucleotides forscreening and for identifing antigenic determinants of the StAR protein(see examples). Peptides having less than about 45 amino acid residuesmay be chemically synthesized by the solid phase method of Merrifield(1963), which reference is specifically incorporated by referenceherein, using an automatic peptide synthesizer with standardt-butoxycarbonyl (t-Boc) chemistry that is well known to one skilled inthis art in light of this disclosure. The amino acid composition of thesynthesized peptides may be determined by amino acid analysis with anautomated amino acid analyzer to confirm that they correspond to theexpected compositions. The purity of the peptides may be determined bysequence analysis or HPLC.

In still another embodiment of the present invention, methods ofpreparing a StAR protein composition are provided. In one aspect, themethod comprises growing recombinant host cells comprising a vector thatencodes a protein which includes an amino acid sequence in accordancewith SEQ ID NO:2 or 18, or that includes a nucleic acid sequence asdefined in SEQ ID NO: 1 or SEQ ID NO: 19, under conditions permittingnucleic acid expression and protein production followed by recoveringthe protein so produced. The host cell, conditions permitting nucleicacid expression, protein production and recovery, will be known to thoseof skill in the art, in light of the present disclosure of the StARgene. A preferred host cell is a COS cell.

Modifications and changes may be made in the sequence of the StARpeptides or protein of the present invention and still obtain a peptideor protein having like or otherwise desirable characteristics. Forexample, certain amino acids may be substituted for other amino acids ina peptide without appreciable loss of function. Since it is theinteractive capacity and nature of an amino acid sequence that definesthe peptide's functional activity, certain amino acid sequences may bechosen (or, of course, its underlying DNA coding sequence) andnevertheless obtain a peptide with like properties. It is thuscontemplated by the inventors that certain changes may be made in thesequence of a StAR peptide or protein (or underlying DNA) withoutappreciable loss of its ability to fimction.

Substitution of like amino acids can be made on the basis ofhydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein byreference, states that the following hydrophilicity values have beenassigned to amino acid residues: arginine (+3.0); lysine (+3.0);aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine(+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5+1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine(−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine(−2.3); phenylalanine (−2.5); tryptophan (−3.4). It is understood thatan amino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent peptide.In such changes, the substitution of amino acids whose hydrophilicityvalues are within +2 is preferred, those which are within +1 are morepreferred, and those within +0.5 are most preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threomine; glutamine and asparagine; and valine,leucine and isoleucine.

Two designations for amino acids are used interchangeably throughoutthis application, as is common practice in the art. Alanine=Ala (A);Arginine=Arg (R); Aspartate=Asp (D); Asparagine=Asn (N); Cysteine=Cys(C); Glutamate=Glu (E); Glutamine=Gln (Q); Glycine =Gly (G);Histidine=His (H); Isoleucine=Ile (1); Leucine=Leu (L); Lysine=Lys (K);Methionine=Met (M; Phenylalanine=Phe (F); Proline=Pro (P); Serine=Ser(S); Threonine=Thr (T); Tryptophan=Trp (W); Tyrosine=Tyr (Y); Valine=Val(V).

While discussion has focused on functionally equivalent polypeptidesarising from amino acid changes, it will be appreciated that thesechanges may be effected by alteration of the encoding DNA, taking intoconsideration also that the genetic code is degenerate and that two ormore codons may code for the same amino acid.

Another aspect of the invention is a method of preparing a recombinantsteroidogenic acute regulatory protein encoded by the purified nucleicacid molecule having a nucleotide sequence of SEQ ID NO:1 or 19, themethod comprising the steps of preparing a recombinant host bearing thenucleic acid molecule, the host being capable of expressing the protein,culturing the recombinant host to produce steroidogenic acute regulatoryprotein, and collecting the recombinant steroidogenic acute regulatoryprotein having an amino acid sequence essentially as set forth in SEQ IDNO:2 or 18. In one aspect, the recombinant host is a COS cell.

A further embodiment of the present invention relates to a purifiednucleic acid molecule encoding StAR protein having an amino acidsequence essentially as set forth in SEQ ID NO: 2, said nucleic acidmolecule obtained by a process of; i) preparing oligonucleotides thatencode a segment of an amino acid sequence of SEQ ID NO:2 and that haveat least about 17 nucleotides; ii) screening an animal cell DNA librarywith said oligonucleotides; and iii) obtaining the purified nucleic acidmolecule encoding StAR protein having an amino acid sequence essentiallyas set forth in SEQ ID NO: 2 or 18.

Pharmaceutical Preparations.

Another aspect of the present invention provides therapeutic agents forthe treatment of steroid hormone-dependent disorders in an animal. Thetherapeutic agent comprises an admixture of StAR peptide or protein in apharmaceutically acceptable excipient. Most preferably, the therapeuticagent will be formulated so as to be suitable for injection.Pharmacologically active peptides of StAR may also be provided to asubject via gene therapy. Many different vehicles exist foraccomplishing this end, such as incorporation of the StAR gene, orfragment thereof, into an adenovirus, retrovirus, or other techniquesknown to those of skill in the art in light of the present disclosure.Ex vivo gene therapy is also contemplated as another mode ofadministration.

Such preparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of the unit. The amount of active compounds in suchtherapeutically usefuil compositions is such that a suitable dosage willbe obtained.

The active compounds may be administered parenterally orintrapertoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial ad antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions. See, for example, Remington(1995), which reference is incorporated by reference herein.

Antibodies.

In another aspect, the present invention includes an antibody that isimmunoreactive with a StAR polypeptide as described for the invention.An antibody can be a polyclonal or a monoclonal antibody. In someembodiments, the antibody is a monoclonal antibody. In addition, theantibodies may comprise recombinant antibodies and may be obtainedemploying the information provided here on StAR in conjunction withthose techniques well know to those of skill in the art. Theseantibodies may further be described as chimeric recombinant antibodies,particularly humanized chimeric antibodies specific for StAR. Means forpreparing and characterizing antibodies are well known in the art (See,e.g., Antibodies “A Laboratory Manual, E. Howell and D. Lane, ColdSpring Harbor Laboratory, 1988). A preferred polyclonal antibody hasbinding specificity for amino acids 1-26, 10-26, 3647, or 88-98 of SEQID NO:2. In addition, the antibodies may comprise recombinantantibodies, and may be obtained employing the information provided hereon StAR in conjunction with those techniques well known to those ofskill in the art. These antibodies may frrher be described as chimericrecombinant antibodies, particularly humanized chimeric antibodiesspecific for StAR. See, for example, Munro (1984) Nature 312:597; U.S.Pat. No. 5,225,599.

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide of the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically an animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster or a guinea pig. Because of the relatively large blood volume ofrabbits, a rabbit is a preferred choice for production of polyclonalantibodies.

Antibodies, both polyclonal and monoclonal, specific for the peptides ofthe present invention may be prepared using conventional immunizationtechniques, as will be generally known to those of skill in the art. Acomposition containing antigenic epitopes of the peptide sequences,isolated peptides, or fragments thereof can be used to immunize one ormore experimental animals, such as a rabbit or mouse, which will thenproceed to produce specific antibodies against StAR. Polyclonal antiseramay be obtained, after allowing time for antibody generation, simply bybleeding the animal and preparing serum samples from the whole blood.

In one embodiment, the polyclonal antibodies to StAR were prepared byinjecting a rabbit with a StAR peptide 88-98 (SEQ ID NO:8) conjugated toKLH (keyhole limpet hemocyanin) mixed with a StAR peptide 88-98 modifiedaccording to the MAP procedure (with a branched lysine core) (See Tam etal (1989) J of Immun. Methods, 123:53-61) incorporated herein byreference. Prior attempts to raise antibody using as immunogen the mouseStAR peptide, 97-107 was less successful generating antibody.

To obtain monoclonal antibodies, one would also initially immunize anexperimental animal, often preferably a mouse, with a purified peptidecomposition. One would then, after a period of time sufficient to allowantibody generation, obtain a population of spleen or lymph cells fromthe animal. The spleen or lymph cells can then be fused with cell lines,such as human or mouse myeloma strains, to produce antibody-secretinghybridomas. These hybridomas may be isolated to obtain individual cloneswhich can then be screened for production of antibody to the desiredpeptide.

Following immunization, spleen cells are removed and fused, using astandard fusion protocol (see, e.g, The Cold Spring Harbor Manual forHybridoma Development, incorporated herein by reference) withplasmacytoma cells to produce hybridomas secreting monoclonal antibodiesagainst the desired peptide. Hybridomas which produce monoclonalantibodies to the selected antigens are identified using standardtechniques, such as ELISA and Western blot methods.

Hybridoma clones can then be cultured in liquid media and the culturesupernatants purified to provide the peptide-specific monoclonalantibodies. In general, monoclonal antibodies to the peptide antigen canbe used in the identification of steroid hormone-dependent disorders. Itis proposed that the monoclonal antibodies of the present invention willfind useful application in standard immunochemical procedures, such asELISA and Western blot methods, as well as other procedures which mayutilize antibody specific to common or allelically distinct peptideepitopes.

Monoclonal and polyclonal antibodies raised against peptides or proteinof the present examples are useful for (1) screening a cDNA expressionlibrary in the process of cloning a gene that encodes a particularprotein or related protein (for example, the SUPERSCREENOimmunoscreening system from AMERSHAM®), (2) facilitating thepurification of a particular protein or related protein by using columnchromatography to which the monoclonal antibody is bound, and (3)providing reagents necessary for a diagnostic immunoassay for screeningbiological samples.

Monoclonal antibodies are obtained using the following procedure:

Immunization Schedule for Raising Monoclonal Antibodies

1. For each mouse, mix 250 izl of antigen solution containing 10 μg ofantigen with 250 μl of complete Freund's adjuvant. Inject six BALB/cfemale mice ip (intraperitoneal injection).

2. After 14 days, repeat the injections of antigen and incompleteFreund's adjuvant.

3. Collect tail bleeds from immunized mice on day 24. Do 1 in 5dilutions in phosphate buffered saline (PBS) and test all samples bycomparison with similar dilutions of normal mouse serum in a dot blot.

4. On day 35, inject all animals ip with antigen and incompleteFreund's.

5. Day 45, do tail bleeds and test by dot blot. All serum sampleschecked by immunoprecipitation against in vivo radiolabeled antigenpreparation.

6. Day 56, inject best responder, 100 μl iv and 100 μl ip. All othersget ip injection with incomplete Freund's.

7. Day 59, fuse splenocytes from best responder.

In still another embodiment of the invention, a hybridoma cell linewhich produces a monoclonal antibody which specifically binds StARprotein is provided. Most particularly, the hybridoma cell line is ananimal hybridoma cell line prepared by a process of immunizing ananimal, such as a mouse or a rat, with StAR protein, isolating anti-StARantibody producing cells from the immunized animal, and fusing theantibody producing cells with a neo-plastic animal cell line to obtain ahybridoma cell line. The resultant hybridoma tissue culture supernatantsare screened for monoclonal antibodies as follows:

1. A protein solution of at least 1 μg/ml of antigen is added to anitrocellulose sheet at 0.1 ml/cm². Allow the protein to bind to thepaper for 1 hr. Higher concentrations of proteins will increase thesignal and make screening faster and easier. If the amount of protein isnot limiting, concentrations of 10-50 μg/ml should be used.Nitrocellulose can bind approximately 100 μg of protein per cm².

2. Wash the nitrocellulose sheet three times in PBS.

3. Place the sheet in a solution of 3% BSA in PBS with 0.02% sodiumazide for 2 hr to overnight. To store the sheet, wash twice in PBS andplace at 4° C. with 0.02% sodium azide. For long-term storage, shake offexcessive moisture from the sheet, cover in plastic wrap, and store at−70° C.

4. Place the wet sheet on a piece of parafihm, and rule with a soft leadpencil in 3-mm squares. Cut off enough paper for the number of assays.

5. Apply 1 μl of the hybridoma tissue culture supernatant to eachsquare. Incubate the nitrocellulose sheet on the parafilm at roomtemperature in a humid atmosphere for 30 min.

Along with dilutions of normal mouse serum, include dilutions of themouse serum from the last test bleed as controls. Dilutions of the testsera are essential to control correctly for the strength of the positivesignals. Mouse sera will often contain numerous antibodies to differentregions of the antigen and therefore will give a stronger signal than amonoclonal antibody. Therefore, dilutions need to be used to lower thesignal. Good monoclonal antibodies will appear 10-fold less potent thangood polyclonal sera.

6. Quickly wash the sheet three times with PBS, then wash two times for5 min each with PBS.

7. Add 50,000 cpm of ¹²⁵I-labeled rabbit anti-mouse immunoglobulin per3-rnu square in 3% BSA/PBS with 0.02% sodium azide (about 2.0 ml/cm²).

8. After 30-60 min of incubation with shaking at room temperature, washextensively with PBS until counts in the wash buffer approach backgroundlevels.

9. Cover in plastic wrap and expose to X-ray film with a screen at −70°C.

The hybridoma identified as producing antibody to the protein ofinterest is passaged as follows:

1. Inject 10⁷ (or less) cells into female mice that have been injectedip about 1 week earlier with 0.5 ml of pristane or incomplete Freund'sadjuvant. These types of injections are also used to prime mice forascites production, and this may serve as a convenient source ofappropriate hosts. If no mice are available, inject mice with incompleteFreund's adjuvant and wait 4 hr to 1 day before injecting the hybridomacells. The animals must be of the same genetic background as the cellline.

2. If an ascites develops, tap the fluid and transfer into a sterilecentrifuge tube.

3. Spin the ascites at 400g for 5 min at room temperature.

4. Remove the supernatant. Resuspend the cell pellet in 10 ml of mediumsupplemented with 10% fetal bovine serum and transfer to a tissueculture plate. The supernatant can be checked for the presence of theantibody and used for further work if needed.

5. Handle as for normal hybridomas, except keep the cells separate fromthe other cultures until there is little chance of the contaminationreappearing.

The present invention in still another aspect defines an immunoassay forthe detection of a StAR protein in a biological sample. In oneparticular embodiment of the immunoassay, the immunoassay comprises;preparing an antibody having binding specificity for StAR protein toprovide an anti-StAR antibody, incubating the anti-StAR antibody withthe biological sample for a sufficient time to permit binding betweenantibody and StAR present in said biological sample, and determining thepresence of bound antibody by contacting the incubate of the sample andantibody with a detectably labeled antibody specific for the anti-StARantibody, wherein the presence of anti-StAR antibody in the biologicalsample is detectable as the measure of the detectably labeled antibodyfrom the biological sample. In some embodiments, the antibody ispreferably a monoclonal antibody having binding specificity for the StARamino acid sequence 88-98 of SEQ ID NO:2 or a region of identical aminoacid sequence in SEQ ID NO. 18 (See FIG. 5).

By way of example, the antibody may be labeled with any of a variety ofdetectable molecular labeling tags. Such include, an enzyme-linkedantibody, a fluorescent-tagged antibody, or a radio-labelled antibody.

A further embodiment of the invention is a method for detecting achromosomal genetic lesion comprising the steps of i) preparing anucleic acid probe having a nucleotide sequence that includes at least a17-base segment of SEQ ID NO:1 or 19 corresponding to a region of a StARgenetic lesion in a diseased patient sample; corresponding to a regionof a StAR genetic lesion in a diseased patient sample; ii) contacting achromosomal sample with the probe to allow hybridization of the sampleto the probe under conditions standard for hybridization fidelity andstability, wherein lack of specific hybridization of the probe and thechromosomal sample provides for detection of a potential genetic lesionin the chromosome. The genetic lesion may be a deletion, arearrangement, an insertion, a transition, a transversion, a frameshift,a missense or a nonsense mutation. In particular, the genetic lesioncorrelates with the presence of lipoid congenital adrenal hyperplasia.Human tissue samples may be biopsy material from adrenal tissue, gonadaltissue, or blood.

In another aspect of the invention, a screening method for lipoidcongenital adrenal hyperplasia is provided. The method comprises thesteps of i) obtaining a chromosomal sample to provide a test sample; ii)preparing a nucleic acid probe having a nucleotide sequence essentiallyas set forth in SEQ ID NO:1 or 19; and, ii;) contacting the test samplewith the nucleic acid probe under hybridization conditions allowing fordetection of a mismatch in a hybridizing molecule as a screening methodfor lipoid congenital adrenal hyperplasia. A mismatch is determined mostreadily by determining the nucleotide sequence of the hybridizingmolecule, a difference in the nucleotide sequence of the hybridizingmolecule and the nucleotide sequence of SEQ ID NO: 1 or 19 provides ascreen for lipoid congenital adrenal hyperplasia.

Further embodiments of the invention include; a method for stimulatingcholesterol transport, a method for increasing production ofprogesterone, and a method for increasing steroidogenesis; these methodscomprise administering a pharmacologically effective amount ofsteroidogenic acute regulatory protein having an amino acid sequenceessentially as set forth in SEQ ID NO:2 or 18. Progesterone is usedclinically in a variety of applications in males and females. Methodsfor providing enhanced production of progesterone are thus a valuableapplication of the StAR compositions of the present invention. Theprotein may be delivered by recombinant means, i.e., synthesis from anexpression vector containing nucleic acid sequences encoding theprotein.

Following long-standing patent law convention, the terms 'a” and “an”mean “one or more” when used in this application, including the claims.

Abbreviations

Bt₂cAMP: N⁶, 2′-O-dibutyryladenosine-3′:5′-cyclic monophosphate

CHAPS: 3-[3-cholamidopropyl dimethylammonio] 1-propanesulfonate

CSCC: cholesterol side-chain cleavage

DBI: diazepam binding inhibitor

hCG: human chorionic gonadotropin

HPLC: high performance liquid chromatography

IOD: integrated optical density

LH: luteinizing hormone

Mops: 3-[N-Morpholino]propane-sulfonic acid

PAGE: polyacrylamide gel electrophoresis

PBR: peripheral benzodiazepine receptor

PBS: Dulbecco's phosphate-buffered saline with calcium and magnesium

PCR: polymerase chain reaction

SAP: steroidogenesis activator polypeptide

SCP₂: sterol carrier protein 2

WAY: Waymouth's MB 752 media

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to fuirther demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Restriction map of the isolated cDNA clone encoding the 37 kDaprecursor protein. The initiating ATG codon and the termination codon,TAA, are indicated by vertical bars along with several uniqueendonuclease restriction sites. The arrowheads indicate the position ofthe degenerate oligonucleotides designed from peptides 25 and 23, andthe hatched bar denotes the region amplified by PCR using theseoligonucleotides. The PCR amplified product was used to screen the cDNAlibrary. Both the coding and noncoding strand of the cDNA weresequenced. The solid lines shown beneath the map represent theoverlapping fragments of the coding strand which were sequenced whilethe dashed lines represent the noncoding strand fragments which weresequenced by the dideoxynucleotide sequencing method of Sanger et al.(1977) using the Sequenase Version 2 kit (United States Biochemical).

FIG. 2. Nucleotide sequence of the 37 kDa cDNA clone (SEQ ID NO: 1) andthe deduced amino acid sequence for the protein (SEQ ID NO:2). Thenucleotides are numbered to the left with position I as the firstnucleotide in the CDNA and the amino acids are numbered to the rightwith amino acid 1 being the initiating methionine of the mitochondrialprecursor. Capital letters are used for the coding region of the cDNAand to denote the in-frame stop codon in the 5′ untranslated region andthe polyadenylation signal and poly (A)+tail in the 3′ untranslatedregion. The amino acids corresponding to peptides 23, 25, and 45 areunderlined and the corresponding peptide is indicated below the line.The underlined nucleotide sequences were included in the set ofdegenerate oligonucleotides designed from the amino acid sequence. Theantibody was raised against amino acids 88-98 which includes most ofpeptide 25. The mature protein begins with the methionine at position 48(see Example 2 regarding cleavage of the precursor protein).

FIG. 3. Nucleic acid sequence of human StAR and mouse StAR. Segments ofhigh identity of nucleic acid sequence are shown.

FIG. 4. Effect of KN93 on agonist-stimulated aldosterone production andStAR protein expression in H295R cells. Cells were incubated for sixhours with Ang II (100 nM), K+(16 μM), and Bay K(1 μM) in the presenceor absence of KN93 (3μM). The medium aldosterone content was determinedby R.I.A. and normalized to the tissue culture well protein content.Data points represent the mean ±SE of values from six separate culturewells expressed as fold increase over basal. Significant inhibitionrelative to the control response is indicated at *p<0.01. StAR proteinwas examined using equivalent amounts of total cellular lysate (30μg) byimmunoblot analysis as described in the Methods. Results arerepresentative of those obtained from three separate experiments.

FIG. 5. Comparison of the deduced amino acid sequences from knownnucleotide sequences of the putative coding region of StAR protein frommouse, human, bovine and ovine. Only a partial sequence is presented forovine. Putative conserved phosphorylation sites that correspond toconsensus motifs are highlighted for PKA and CAM kinase 1 (A), PKC (C),CKII (CK), and P34CDC2 kinase (CD). The proposed cleavage sites for themitochondrial proteases, matrix-processing protease, and mitochondrialintermediate-processing peptide are noted (C1 and C2); however, only themouse sequence strictly follows the consensus motif for two cleavageswith the other sequences predicting one cleavage. Another putative PKAphosphorylation site is found in bovine and human sequences at position277. While Ser 277 is not conserved in the murine sequence, there is aSer at 278. If phosphorylation is crucial to the flunction of StARprotein and this Ser is the residue that is phosphorylated, then thismay explain how the C-terminal deletion mutants found in lipoid CHpatients affect StAR fimction.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have utilized the MA-10 mouse Leydig tumor cellline to study the acute regulation of steroidogenesis. Specifically, theinventors have identified a family of hormone-induced mitochondrialproteins in MA-10 cells that regulate cholesterol delivery to the innermitochondrial membrane and the CSCC. These proteins have been describedas the mitochondrial 37 kDa, 32 kDa, and 30 kDa molecular weightproteins and they are synthesized in response to either LH and HCG or bystimulation with the CAMP analogue, Bt2cAMP. The 30 kDa species consistsof four separate proteins and proteolytic digestion of all four formsindicates that they are all modified forms of the same protein. Pulsechase experiments and tryptic peptide mapping of the 37 kDa and 30 kDaproteins indicated that the 37 kDa form is a precursor to the 30 kDaprotein.

The following data support the involvement of these proteins in theacute regulation of steroidogenesis: i) their synthesis is directlycorrelated to the capacity of the MA-10 cells to produce steroid inresponse to hormone stimulation in both a time and dose responsivemanner; ii) their synthesis is sensitive to cycloheximide; iii) the 30kDa proteins are localized to the mitochondria and are processed from alarger precursor protein of 37 kDa; iv) a rat Leydig tumor cell line(R₂C) which constitutively produces steroids constitutively expressesthe 30 kDa proteins; and v) inhibition of steroidogenesis is concomitantwith inhibition of synthesis of 30 kDa proteins (Stocco and Kilgore,1988; Stocco and Chaudhary, 1990; Stocco and Chen, 1991; Stocco andSodeman, 1991; Stocco, 1992; Stocco and Ascoli, 1993; Stocco et al.,1993; Stocco and Clark, 1993).

The present inventors have now cloned the cDNA for this family ofproteins, and have also more directly determined their fuinction in theregulation of steroid production, particularly in MA-10 cells.

The following examples describe the purification of the MA-10 30 kDaproteins and the isolation and characterization of a fuill length cDNAclone. The cDNA encodes a novel mouse protein of 31.6 kDa, which relatesto the previously described 37 kDa precursor protein of the LH-inducedfamily of mitochondrial proteins. The amino acid sequence at the aminoterminus has been identified by the present inventors to becharacteristic of a mitochondrial targeting signal. Using an in vitrotranscription/translation system, the precursor protein was processedand modified to all forms of the 30 kDa proteins by isolatedmitochondria. Immunoblot analysis of mitochondria isolated from eitherBt₂cAMP-stimulated MA-10 cells or MA-10 cells transfected with the cDNAconfirmed the cDNA encodes the same immunospecific 30 kDa protein. Inaddition, in the absence of hormone stimulation, expression of the 30kDa protein in MA-10 cells resulted in a 1.5-3.5 increase inprogesterone production above cells transfected with vector alone. Thus,the present inventors have demonstrated for the first time thatexpression of the LH-inducible 30 kDa protein directly results in anincrease in steroid biosynthesis. This protein is required in the acuteregulation of hormone-induced steroidogenesis.

Materials & Methods. The materials and methods used in the followingexamples are provided here. In light of these teachings, one of skill inthe art would realize that other equivalent materials and methods may beused in the present invention.

Chemicals. Waymouth's MB 752 medium, Dulbecco's modified Eagle's medium,horse serum, fetal bovine serum, antibiotics, and PBS were purchasedfrom Life Technologies, Inc. (Gaithersburg, MD). Bt₂cAMP, leupeptin,aprotinin, phenylmethylsufonyl fluoride, formaldehyde, and Escherichiacoli alkaline phosphatase were obtained from Sigma (St. Louis, Mo.).Silver nitrate was from Fisher (Houston, Tex.). The ampholines and stocksolutions of nucleic acids were purchased from Pharmacia Biotech Inc.(Piscataway, N.J.). Restriction endonucleases, T7 RNA polymerase,RNAsin, and Taq DNA polymerase were purchased from Promega (Madison,Wisc.). Radiolabeled nucleotides [³²-P] CTP and [³⁵S]-methionine wereobtained from Du Pont NEN (Boston, Mass.). Oligonucleotides weresynthesized and purified by Midland Certified Reagent Co. (Midland,Tex.).

Maintenance of MA-10 and COS 1 cells. The MA-10 mouse Leydig tumor cellline was from Dr. M. Ascoli (Dept. of Pharmacology, Univ. of IowaCollege of Medicine, Iowa City, Iowa). These cells were derived from theM5480P tumor, they have functional LH/CG receptors and produce largeamounts of progesterone rather than testosterone in response to hormonestimulation. The cells were grown in Waymouth's MB/752 media containing15% horse serum (WAY+) at 37 cC in a humid atmosphere under 5% CO₂ andmaintained in culture using standard techniques (Ascoli, 1981). COS 1cells were obtained from the American Type Culture Collection (#CRL1650) and grown in Dulbecco's modified Eagle's medium high glucose mediacontaining 10% fetal bovine serum and 100 units of penicillin/ml and 10units of streptomycin sulfate/ml.

Isolation of Mitochondria. MA-10 cells were stimulated for 6 h with 1 nMBt₂cAMP in Waymouth's media containing 5% horse serum, then washed oncewith PBS (Life Technologies, Inc.) and collected in 0.25 M sucrose, 10mM Tris, pH 7.4, 0.1 mM EDTA by scraping with a rubber policeman. Thecells were lysed by homogenization at 1000 rpm for 25 passes with aPotter Elvehjem homogenizer fitted with a serrated Teflon pestle. Thehomogenate was centrifuged at 600×g for 30 min, and the resultantsupernatant was centrifuged at 9000×g for 30 min to pellet themitochondria. The mitochondrial pellets were stored frozen at −80° C.until used to purify the 30-kDa proteins.

For the in vitro translation reactions, mitochondria were isolated asabove with the following exceptions; MA-1O cells were not stimulatedwith hormone, and the cells were lysed using a glass-on-glass Douncehomogenizer with fitted pestle and homogenized by hand for 25 passes.The mitochondrial pellet was washed once with import buffer [3% bovineserum albumin, 70 mM KC], 220 mM sucrose, 10 mM Mops/KOH (pH 7.2), 2.5mM MgCl₂ (Hartl, 1986) then resuspended in 200 μl of the import bufferto a protein concentration of 7.5 mg/ml. Mitochondria were usedimmediately after isolation for the in vitro translation reaction.Isolation of mitochondria from COS 1 cells for immunoblot analysis wasas described (Clark and Waterman, 1991).

Purification ofthe 30-kDa Proteins from Isolated Mitochondria Ingeneral, mitoplasts (mitochondria stripped of outer membrane) werepurified from isolated mitochondria and solubilzzed with CHAPSdetergent. Preparative one-dimensional SDS-PAGE gels were used toisolate CHAPS soluble proteins of 28-32 kDa in size (30-kDa fraction),and the 30-kDa proteins were isolated and recovered by two-dimensionalSDS-PAGE of the 30-kDa fraction. A more detailed description of thispurification is as follows.

Preparation ofMitoplasts and CHAPS Solubilization. Mitoplasts wereprepared using the methods detailed by Ardail et al. (1991).Mitochondria were resuspended in swelling buffer (10 mM potassiumphosphate, pH 7.4, 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.1mg leupeptin/ml, and 0.04 units aprotinin/ml) to a protein concentrationof 2.5 mg/ml, incubated on ice for 20 min, then homogenized 5 times byhand using a glass Dounce homogenizer fitted with a Teflon pestle. Anequal volume of swelling buffer containing 30% sucrose was added to theshocked mitochondria and mixed thoroughly. Mitoplasts were recovered bycentrifugation at 12,000×g for 30 min, resuspended in swelling buffer toa protein concentration of 2.5 mg/ml, and solubilized by adding a freshsolution of swelling buffer containing 25% CHAPS to achieve a 1:1 mgprotein to mg detergent ratio (0.25% CHAPS final concentration). TheCHAPS soluble sample was recovered after a 100,000×g centrifugation for45 min and concentrated under nitrogen pressure using a Filtronstir-cell with 10,000 molecular weight cut off (Pharmacia Biotech Inc.).Protein was determined for each fraction by the method of Bradford(1976).

Preparative One-dimensional SDS-Polyacrylamide Gel Electrophoresis TheCHAPS soluble sample was separated on a preparative 1.5 mm 12.5%SDS-polyacrylamide gel (Laemmli, 1970). A 5 mm section of thepolyacrylamide gel corresponding to 28-32 kDa band was excised, and theproteins were electroeluted from the gel and concentrated using theCentrilutor microelectroeluter system (Amicon). The position of the 5 mmstrip was determined by ruling a reference lane containing molecularweight markers and cutting the lane from the gel and staining themarkers with Coomassie blue.

Two-Dimensional Polyacrylamide Gel Electrophoresis. Approximately150-250 μg of the 30-kDa fraction (the concentrated 28-32 kDa proteinsfrom the one-dimensional gel) was resolved by two-dimensional PAGE (0°Farrell, 1975), and the proteins were electrophoretically transferred tonitrocellulose in 20 mM Tris/CI pH 7.4, 150 mM glycine, 10%β-mercaptoethanol, 0.01% SDS for 4 h at 350 mA (Deutscher, 1990; Towbin,et al. 1979). The nitrocellulose was transiently stained with Ponseau S(0.2% in 1% acetic acid) to visualize and isolate the specific 30 kDaproteins for subsequent amino acid microsequence analysis. The filterswere destained with 1% acetic acid, and washed thoroughly with HPLCgrade water. The nitrocellulose spots were stored damp at −80° C. untilshipped to the Harvard Microchemical facility where the in situdigestion, peptide separation, and microsequence analysis was performedon a fee-for service basis.

Quantitation of Silver Stained Proteins. The two-dimensional SDS-PAGEgels were fixed in 50% methanol, 12% acetic acid for 1 h, then washedwith 50% EtOH 3 times for 20 min with each wash. To silver stain theproteins, the gel was pretreated with 0.02% sodium thiosulfate for 1min, rinsed 3 times with H₂O for 20 s each rinse, then treated with 0.2%silver nitrate, 0.02% formaldehyde for 20 min. After the silver nitrateimpregnation, the gel was rinsed with H₂O twice and developed with 6%sodium carbonate, 0.02% formaldehyde. The silver-stained image wascaptured, and the integrated optical densities (IOD) of the proteinswere quantitated using the BioImage Visage 2000 computer-assisted imageanalysis system (Biolmage, Ann Arbor, Mich.). The percent of the totalIOD of each spot (protein) was determined automatically and used toquantitate the 30-kDa protein. For example, the percent of total IOD for30-kDa protein 1×mg total protein loaded onto the first dimension gel/mgtotal protein for the fraction=mg 30-kDa protein 1 in that fraction.

Preparation of the Bt2cAMP-stimulated MA-10 Mouse Leydig Tumor Cell cDNAlibrary. Total RNA was isolated from 6-h Bt₂cAMP-stimulated MA-10 cellsby a one-step extraction adapted from the methods of Chomczynski andSacchi (1987) using RNA STAT-60 (Tel-Test B, Inc., Friendswood, Tex.).Poly A⁺ MRNA was twice selected on a gravity flow oligo-dT column (5Prime-3 Prime, Inc.). A λgt22A cDNA library was constructed from thepolyA⁺RNA using the SuperScript Lambda System for cDNA synthesis and λcloning (Life Technologies, Inc.). Briefly, first strand synthesis wasgenerated using a Noll Primer-Adapter and SuperScript RT, an engineeredMoloney murine leukemia virus reverse transcriptase. Second strandsynthesis was generated by nick translation replacement of the templatemRNA and a Sall adapter was ligated to the cDNA ends. The cDNAs weredigested with Noti and Sall restriction enzyres and cloned into λgt22A.The DNA was packaged in vitro using the λ Packaging System (LifeTechnologies, Inc.) and the recombinant phages were stored in 50 mMTris/Cl pH 7.5, 100 MM NaCI, 1 mM MgSO₄, 0.01% gelatin and CHCl₃ at 4°C. The cDNA library contained 9×10⁵ independent clones. The E. colistrain YI09Or (Life Technologies, Inc.) was infected with the stockphage solution, and the library was amplified to a titer of 2×10¹⁰plaque-forming units/ml before the initial screen.

Cloning the 30 kDa CDNA. Standard methods were used to purifybacteriophage X particle from the amplified cDNA library and to extractthe recombinant DNAs (Sambrook, et al., 1989). Degenerativeoligonucleotides were designed based on the amino acid sequences frompeptide 23 and peptide 25 and used for primerdirected amplification ofthe DNA isolated from the library. Since the position of the peptidesrelative to each other within the protein was not known, both the codingand reverse complement sequences were synthesized. The coding andreverse complement sequences for peptide 23 used were 5′ -GCN GAR CAYGGN CCN ACN TGY ATGOG-3′ (SEQ ID NO:9) and 5′ -C CAT RCA NGT NGG NCC RTGYTC NGC-3′ (SEQ ID NO:10), respectively, and for peptide 25 were 5′ -AAY CAR CAR GGN TGG AA-3′ (SEQ ID NO:11) and 5′-TTC CAN CCY TCYTGRTT-3′(SEQID NO:12), respectively. In these designations, N is inosine, R is Aor G, Y is T or C. The corresponding sequences are underlined in FIG. 2.The oligonucleotides were synthesized and purified by HPLC by MidlandCertified Reagent Co. (Mdland, Tex.). Conditions for amplification ofthe DNA by the polymerase chain reaction were 20 mM Tris/CI pH 8.4, 50mM KCI, 2.5 mM MgCl₂, 0.1 mg/ml bovine serum albumin, 10 mMconcentration of each dATP, dCTP,dTTP, dGTP, 50 pmol of each primer, 2.5units Taq DNA polymerase, and 1 μg cDNA (Saiki, et al., 1988). Thirtycycles of amplification were performed at 92° C. for 1 min, 45° C for 30s, and 72° C. for 30 s. A specific PCR product of 400 base pairs wasamplified from the isolated cDNAs and subcloned into the Smal site ofBluescript KS- (Stratagene, La Jolla, Calif.). An [α³²P]-CTP-labeledriboprobe was synthesized and used to screen the cDNA library. Solutionhybridization and stringent washing was performed using standardprocedures (Sambrook, et al., 1989). One positive clone wasplaque-purified from an initial screen of 1×10⁶ clones. The cDNA wasdirectionally subcloned into the prokaryotic expression vector, pSPORT 1(Life Technologies, Inc.), using the Sall and NotI cloning sites. Aseries of nested deletions were constructed (Erase-A-Base kit, Promega,Madison, Wisc.) to generate overlapping clones from both the 5′ and 3′ends of the cDNA. Both strands of the cDNA were sequenced by thedideoxynucleotide sequencing method of Sanger using the Sequenase KitVersion 2 (United States Biochemical Corp., Cleveland, Ohio) (Sanger, etal, 1977). Electrophoresis was performed in an 8% polyacrylamide gel(Hydrolink, AT Biochem, Malvern, Pa.) with 8 M urea and 25% formamide(v/v). The regions sequenced are indicated in FIG. 1.

In vitro Transcription/Translation of the Cloned cDNA. NotI linearizedpSPORT I/cDNA template (2.5,ug) was transcribed in a 100 μl reactioncontaining 40 mM Tris/Cl (pH 7.5), 6 mM MgCl₂, 2 mM spermidine, 10 mMNaCl, 10 mM dithiothreitol, 100 units RNAsin, 0.5 mM of each UTP, CTP,GT?, ATP, and 40 units T7 RNA polymerase for 2 h at 37° C. In vitrotranslation reactions were performed in parallel and included either 15μg of isolated mitochondria alone or mitochondria plus 4 μg of thetranscribed RNA. A rabbit reticulocyte lysate kit was used following theinstructions of the manufacturer (Du Pont-NEN, Boston, Mass.). Theproteins were synthesized in the presence of [³⁵S]-methionine for 1 h at37° C. and the reactions were frozen at −20° C. An equal volume of eachreaction was analyzed by two-dimensional SDS-PAGE as described above.The gels were prepared for fluorography using Resolution (E.M. Corp.,Chestnut Hill, Mass.), dried under moderate heat and vacuum, and exposedto x-ray film at −80° C.

Expression ofthe 30-kDa Protein in A-1 Cells and COS I Cells. The fulllength SalI-NotI 1456 base pair 30-kDa cDNA was subdloned into theeukaryotic expression vector, pCMV (Andersson, et al, 1989). MA-10 cellswere transfected with DNA by a liposome-mediated uptake using theLipofectAMINE reagent (Life Technologies, Inc., Gaithersburg, Md.)(Hawley-Nelson, et al., 1993). Plasmid DNA used in transfectionexperiments was purified by CsCl density gradient followed bypolyethylene glycol precipitation. The DNA was mixed with {fraction(1/10)} the final volume of Waymouth's media minus serum and minusantibiotics (WAY-) and added to an equal volume of WAY- media containingthe LipofectAMiNE reagent. The DNA/lipid solution was gently mixed andincubated for 30 min at room temperature, then WAY- media was added toachieve the final concentration of the DNA and LipofectAMINE reagent of5 μg/ml and 20 μg/ml respectively. The cells were washed once with WAY-media, incubated with transfection mix for 6 h, washed once with PBS,then incubated with Waymouth's plus 15% horse serum. The same procedurewas used for transfection of COS 1 cells except Dulbecco's modifiedEagle's media minus serum and antibiotics was used for the transfectionmedia For isolation of mitochondria for immunoblot analysis, cells weregrown on 100-mm dishes. For progesterone production assays, MA-10 cellswere plated into 96 well plates at 75,000 cells/well the day before theexperiment. Typically, 8 wells each were transfected with either pCMV orpCMV+30-kDa cDNA for one experimental set.

A reporter construct expressing a tartrate-resistant acid phosphatase(TRAP) was used to determine the efficiency of transfection of MA-10cells by the LipofectMINE reagent. The tartrate-resistant acidphosphatase expression plasmid contains the full-length human cDNAcloned into the pcDNAl vector provided by Dr. G. D. Roodman (Univ. ofTexas HSC, San Antonio, Tex.) (Reddy, et al., 1993). Forty-eight hourspost-transfection the cells were fixed directly in the wells with 2%glutaraldehyde, then stained for tartrate-resistant acid phosphataseactivity using an acid phosphatase staining kit (Sigma Chemical Co., St.Louis, Mo.). Several wells of MA-10 cells were transfected with thetartate-resistant acid phosphatase expression plasmid for eachexperiment and the positively stain cells were counted by visualinspection using an inverted light microscope. Typically, 5-7% of thecells were stained positive for tartrate-resistant acid phosphataseexpression.

Immunoblot Analysis. Mitochondria were isolated from either MA-10 or COS1 cells transfected with either pCMV or pCMV+30 kDa cDNA 48 hourspost-transfection. For experiments in which progesterone production wasmeasured (as described above), cells were solubilized directly in thewell with 0.1% SDS, the cell homogenates were collected and combinedfrom all 8 wells, and the protein was precipitated using trichloroaceticacid. The protein was solubilized in sample buffer (25 mM Tris/Cl, pH6.8, 1% SDS, 5% β-mercaptoethanol, 1 mM EDTA, 4% glycerol, and 0.01%bromophenol blue) and loaded onto a 12.5% SDS-PAGE minigel (Mini-ProteanII System, Bio-Rad, Richmond, Calif.). Electrophoresis was performed at200 V for 45 min using standard SDS-PAGE buffer as described above, andthe proteins were electrophoretically transferred to a polyvinylidenedifluoride membrane (Bio-Rad) at 100 V for 2 h at 4° C. using thetransfer buffer described above. For immunodetection of the 30 kDaprotein, antipeptide antibodies were generated in rabbits against aminoacids 88-98 of the 30 kDa proteins (FIG. 2). The peptide was synthesizedand the antibodies were produced in rabbits on a fee for service basisby Research Genetics (Huntsville, Ala.). The immunoblot procedure was asfollows; the membrane was incubated in blocking buffer (PBS buffercontaining 4% Carnation non-fat dry milk and 0.5% Tween-20) at roomtemperature for 1 h, then incubated in fresh blocking buffer containingthe primary label (rabbit sera containing the specific antipeptideantibodies) for an additional hour at room temperature. Next, themembrane was washed with PBS containing 0.5% Tween-20, 3 times for 10min each wash, then incubated for 1 h at room temperature with freshblocking buffer containing the secondary antibody, donkey anti-rabbitIgG conjugated with horseradish peroxidase (Amersham Life Sciences,Arlington Heights, Ill.). The membrane was washed as before, and thespecific signal was detected by chemiluminescence using the Renaissancekit from Du Pont-NEN.

Polyclonal antiserum has been generated to presequences of StAR, inparticular, to a signal sequence from amino acid 10 to 26 of SEQ IDNO:2, and to a targeting sequence from amino acid 36 to 47 of SEQ IDNO:2. Antibodies of these polyclonal antisera were tested andimmunoreact with precursor forms of STAR. Further polyclonal antiserumwas generated to signal sequence from amino acid 1 to 26 of SEQ ID NO:2,this antiserum reacts with the signal peptide.

Radioimmunoassay. 48 hours post-transfection, the growth medium wasreplaced with Waymouth's minus horse serum. After 6 h at 37° C., 5% CO₂,progesterone was measured directly in the media by radioimmunoassay aspreviously described (Resko, et al, 1974). The progesterone antibody wasobtained from Holly Hill Biologicals, (Hillsboro, Oreg.).

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Purification of 30-kla Proteins 1 and 2

The present example provides for the purification of the 30 kDa proteins1 and 2. The family of LH-inducible proteins previously identified andcharacterized by the present inventors in mitochondria isolated fromMA-10 cells represented approximately 0.2% of the total cell protein and0.7% of the mitochondrial protein (Table 3). Due to the limited amountof the in vivo induced protein, the present inventors purified the 30kDa proteins from the mitochondria by enriching a postmitochondrialfraction for the specific 30 kDa proteins and separating the enrichedfraction by two-dimensional SDS-PAGE. The proteins wereelectrophoretically transferred to nitrocellulose, and the specific 30kDa proteins were isolated by excising the nitrocellulose spotcontaining the bound protein. The purification scheme developed toisolate the 30 kDa proteins is summarized under “Materials and Methods”hereinabove. The objective was to isolate a sufficient amount of the 30kDa protein for in situ tryptic digestion, peptide separation, andmicrosequence analysis.

Proteolytic digestion of each of the 30 kDa forms produced identicalpeptides, indicating that the difference in these four forms is due topost-translational modifications of a single protein. The presentinventors had previously shown that the difference in the isoelectricpoints for forms 3 and 4 is due to phosphorylation of forms 1 and 2. The30 kDa mitochondrial proteins are processed from a larger precursorprotein synthesized in response to hormone stimulation of MA-10 cells.However, the precursor protein has a short half-life and is difficult todetect in MA-10 cells (Stocco and Sodeman, 1991; Epstein andOrme-Johnson, 1991). For this reason the present inventors focused onthe purification of the 30Mka proteins which are stable in mitochondriaisolated from MA-10 cells. These proteins had been detected bymetabolically labeling MA-10 cells in vivo with[³⁵S]-methionine/cysteine and isolating the mitochondria for twodimensional SDS-PAGE analysis followed by fluorography.

The present inventors first determined if the quantity of the 30 kDaproteins in isolated mitochondria was sufficient to detect by proteinstaining. The 30k)a proteins were readily detectable by silver-stainafter two dimensional SDS PAGE of mitochondria isolated fromBt₂cAMP-stimulated MA-10 cells. As expected, the proteins were absent inunstimulated cells. The major difficulty in the purification of the 30kDa proteins from MA-10 cells was the lack of a bioassay, therefore,each step of the purification was monitored by silver-stained twodimensional SDS PAGE analysis of the proteins.

Table 3 summarizes the enrichment and recovery of the 30 kDa proteins.

TABLE 3 Purification of the 30-kDa Proteins from Bt₂cAMP-stimulatedMA-10 Cells¹ Enrichment of the 30-kDa proteins μg 30-kDa proteins/ %recovery Total protein mg sample -fold 30-kDa Fraction % recoveryprotein enrichment proteins Mitochondria 100  7 1 100 Mitoplasts 56 ± 10ND^(a) ND ND CHAPS-soluble 22 ± 6  18 2.6 55 30-kDa fraction 0.6 ± 0.394 ± 28 13 7 n = 16 ¹Shown are the results from the purification of the30-kDa proteins from 40-50 mg of mitochondria. Each fraction wasseparated by two dimensional-gel electrophoresis and the proteins werevisualized by silver stain. 1 gel was analyzed for the mitochondrialfraction, 2 gels with different protein concentrations for theCHAPS-soluble fraction, # and 4 gels with varying protein concentrationsfor the 30-kDa fraction were run simultaneously and stained for protein.The silver-stained image was captured, and the integrated intensity (II)of each spot (protein) was estimated using a BioImage Visage 2000(Millipore). The percent of the total integrated intensity of eachspot/protein # was automatically determined. To estimate the amount of30-kDa protein, the percent of total II for the 30kDa protein spots weremultiplied by the amount of total protein loaded onto the firstdimension gel. The results are shown as micrograms 30-kDa protein (sumof 1-4) per mg of fraction sample. ^(a)ND, mitoplast fraction notdetermined.

The purification achieved a 13-fold enrichment with a 7% recovery of the30 kDa proteins. The goal was to sufficiently enrich the 30 kDa proteinsin a final fraction in order to resolve a sufficient quantity (1-2 Ag)of the specific 30 kDa proteins by 2D SDS-PAGE. The ID preparative gelenriched the 30 kDa proteins to approximately 100 μg/mg of the final 30kDa fraction which allowed the present inventors to isolate the 30 kDaprotein. The 30 kDa fraction was treated with alkaline phosphatase justprior to 2D SDS-PAGE to concentrate the 30 kDa proteins into forms 1 and2 (Stocco and Clark, 1993). Comparison of the protein profiles for the30 kDa fraction purified from mitochondria isolated from control andBt₂cAMP-stimulated MA-10 cells was used to verify that the correctprotein spots, 30 kDa 1 and 2, were isolated. Using this purification,approximately 75 mg of isolated mitochondria was required to isolateapproximately 200 pmol of the 30 kDa proteins from Bt2cAMP-stimulatedMA-10 cells. Quantitatively, 60% of the total 30 kDa proteins wasrecovered in 30 kDa 2, and 40% was recovered in 30 kDa 1. The differencebetween form 1 and form 2 may be that of methylation, acetylation,sulfation, prenylation, or myristylation, and the like.

EXAMPLE 2 Cloning the CDNA and Analysis of the Encoded 30 kDa Protein

The present example provides for the cloning and sequence analysis ofthe cDNA encoding the 30 kDa protein, and analysis of the proteinsequence. Even though 30kDa 1 and 30 kDa 2 were thought to be identicalproteins, they were isolated and stored separately. To ensure onehomogeneous protein was used for microsequence analysis, only the 30 kDaprotein 2 was sent to the Harvard Microchemical Facility where in situdigestion, tryptic peptide separation, and microsequence analysis wasperformed on a fee-for-service basis. Three tryptic peptides, #23, #25,and #45, were selected for microsequence analysis. The amino acidsequences for the peptides were determined to be:

Peptide 23 : AEHGPTCMVLHPLA, (SEQ ID NO:3)

Peptide 25 : ALGILNNQEGWK, (SEQ ID NO:4)

Peptide 45 : GSTCVLAGMATIHFGEMPEQ, (SEQ ID NO:5)

The GenEMBL and SWISS-PROT data bases were searched for similarities tothe three peptide sequences and no significant homologies were found(Fasta and TFasta programs, GCG Package, University of Wisconsin,Madison Wisc.).

A cDNA library was constructed using polyA+RNA purified from total RNAthat was isolated from Bt2cAMP-stimulated MA-10 cells as describedhereinabove. Using the amino acid sequences of the 3 peptides,degenerative oligonucleotides 17-24 bases in length were synthesized andused to amplify the 30 kDa cDNA from the cDNA library by the polymerasechain reaction (PCR)(Saiki et al., 1988). A 400bp specific PCR productwas amplified using a combination of the peptide 25 coding and peptide23 reverse complement oligonucleotides. The PCR generated DNA was usedto probe the cDNA library and a 1456 bp full-ength clone was isolated.Both strands of the cDNA were sequenced and a partial restriction map isshown in FIG. 1. Also included are the positions of the PCR amplifiedsequence, the initiating ATG codon, and the termination TAA codon. FIG.2 shows the nucleotide sequence of the 30 kDa cDNA which contains anopen reading frame of 852 base pairs that encodes a protein of 284 aminoacids with a calculated molecular weight of 31.6 kDa. The deduced aminoacid sequence for the 30 kDa protein is shown by three letter code underthe nucleic acid sequence. The three peptide sequences derived from theprotein microsequence analysis are encoded in the cDNA (the amino acidsare underlined in FIG. 2) which confirmed the translation reading frame.

Although the predicted molecular weight based on the deduced amino acidsequence is lower than the observed size of the mitochondrial precursorprotein by 2D SDS-PAGE, inspection of the amino terminal amino acidsequence for the deduced protein revealed characteristics consistentwith mitochondrial targeting sequences (von Heijne, 1986; von Heijne etal., 1989). Namely, the first 25 amino acids lack acidic amino acids,are enriched in Arg (12%), Ser (8%), Ala (8%), and Leu (12%), and thepredicted secondary structure is an amphipathic alpha helix. Inaddition, the amino acids at positions 38, 40, and 43 are Arg, Leu, andSer, respectively, which would fit the amino acid consensus cleavagesite, R-X-O-X-X-S, where X represents any amino acid and 4 represents ahydrophobic residue (Hendrick, et al., 1989). This amino acid motif ishighly conserved in mitochondrial presequences that undergo a 2 stepsequential cleavage of mitochondrial precursors by the matrix processingprotease (MPP) and the mitochondrial intermediate processing peptide(MIP) (Kalousek, et al., 1988; Kiebler et al., 1993). No significantsimilarities were found to the cDNA sequence when the GenEMBL andSWISS-PROT data bases were searched.

The signal sequence is represented by amino acids at positions 1 toabout 26 of SEQ ID NO:2, more particularly, from about amino acids atpositions 10 to 26 of SEQ ID NO:2. The targeting sequence is representedby amino acids at about positions 36 to 47 of SEQ ID NO:2. The mature 30kDa protein has methionine at position 48 as the N-terminal amino acid.

To confirm that the cDNA clone encodes the precursor and maturemitochondrial proteins, the cDNA was transcribed in vitro and thesynthesized RNA was used in an in vitro translation reaction. A twodimensional SDS-PAGE of the [³⁵S]-methionine labeled in vitro translatedproteins in the presence of mitochondria demonstrated that the mobilityof the proteins was identical to the LH-induced newly synthesizedproteins in MA-10 cells which were previously identified as the 37 kDaprecursor protein and the 30 kDa mitochondrial proteins. Therefore, thecDNA obtained based on the amino acid sequence data for the 30 kDa 2protein encodes all forms of the previously described family ofmitochondrial proteins.

EXAMPLE 3 Improved Production of Progesterone and StAR protein byRecombinant Means; Induction of Steroidogenesis

The present example demonstrates that the expression of the 37 kDaprotein has an effect on steroid production in mammalian cells.

MA-10 cells were transfected with the 30 kDa cDNA and progesteroneproduction was measured as follows. The full-length cDNA was subclonedinto the pCMV eukaryotic expression vector and transfected into MA-10cells using LipofectAMINE (Life Technologies, Inc.). Cells (75,000 perwell) were plated in a 96 well plate the day before transfection. Thecells were incubated with the DNA/lipid transfection mixture for 6hours, washed one time with PBS, and then incubated in Waymouths +15%horse serum. Forty-eight hours post-treisfection the cells were washedwith PBS and Waymouths media (minus serum) was placed back onto thecells. After 6 h, the medium was removed and progesterone was measuredby radioimmunoassay. The cells were lysed directly in the wells with0.1% SDS and protein was determined by the method of Bradford (1976).Progesterone production is shown in Table 4 as picograms progesteroneproduced per mg protein per 6 h. The tansfected cells were not treatedwith hormone and progesterone was measured directly in the media after a6 h incubation. A significant increase in progesterone production wasobserved in MA-10 cells transfected with the 30 kDa cDNA compared tocells transfected with the PCMV vector alone (Table 4). Expression ofthe 30 kDa protein resulted in a 1.5 to 3.7 fold increase insteroidogenesis with an average rate of 166 pg progesterone produced permg protein per hour.

TABLE 4 Progesterone production in MA-10 cells transiently transfectedwith the 30kDa cDNA¹ pCMV + pCMV cDNA/ Control pg progesterone/ pCMV(nontrans- mg protein/6 pCMV + -fold Study² n fected) h cDNA increase² I3  ND³ 361 ± 38  1239 ± 347 3.4 II 4 ND 317 ± 77  519 ± 42 1.6 III 8 ND403 ± 145 1775 ± 444 3.7 IV 8 756 ± 135 787 ± 174 1148 ± 174 1.5 V 8 469± 58  428 ± 81  1378 ± 233 3.2 VI 8 779 ± 171 1071 ± 143  3146 ± 768 2.9¹Control (non-transfected) cells were grown in WAY+ for 48 h and washedonce with PBS+ before WAY− media was added; pCMV, MA-10 cells weretransfected with the pCMV vector alone; pCMV + cDNA, MA-10 cells weretransfected with the pCMV + 30kDa cDNA. n represents the number of wellstransfected for each experiment. The progesterone was measured in eachwell and the mean ± the standard deviation is shown for each study. TheStudent's t test was used to # determine the statistical differencebetween the pCMV and pCMV + 30kDa cDNA samples. ²In every study, thedifference was significant with a p value <0.01. ³ND, not determined.

The level of expression of the 30 kDa protein in the transfected MA-10cells was determined by immunoblot analysis. Antibodies having bindingspecificity for amino acids 88-98 of the 30 kDa proteins (see Materialsand Methods) recognized a protein at approximately 30 kDa only inmitochondria isolated from Bt₂cAMP-stimulated MA-10 cells while noimmunodetectable protein was observed in the non-stimulated cells. Inaddition, the antibody recognized all four 30-kDa protein spotsspecifically when mitochondrial proteins from stimulated MA-10 cellswere resolved by two-dimensional SDS-PAGE. Cell homogenates of the MA-10cells that had been transfected with pCMV +30 kDa cDNA and used forprogesterone production for expression of the 30 kDa protein weretested, however, no immunodetectable protein was observed. Since thepCMV vector does not replicate in MA-10 cells, and only 5% of the cellsare transfected with plasmid DNA based upon expression of thetartrate-resistant acid reporter protein, it was not surprising that theprotein could not be detected in cell homogenates collected fromapproximately 1×10⁶ cells (8 wells from a 96 well plate).

In order to verify that the cDNA was being expressed in eukaryoticcells, COS 1 cells were used to transfect the pCMV +30 kDa cDNA sincethe pCMV plasmid can be replicated in these cells and approximately 80%of the cells are transfected with plasmid DNA based on the expression ofreporter protein. Forty-eight hours post-transfection, mitochondria wereisolated and the 30 kDa protein expression was determined by immunoblotanalysis. An immunospecific protein of approximately 30 kDa was readilydetectable in isolated mitochondria only from COS 1 cells that had beentransfected with the 30 kDa cDNA, indicating the cDNA does express thesame protein as the Bt₂cAMP-stimulated MA-10 cells. In addition, a 36.5kDa protein was detected in the COS 1 cells which would be consistentwith the precursor protein. Importantly, the 30 kDa protein was alsodetectable in MA-10 cells transfected with PCMV +30 kDa cDNA byimmunoblot analysis when isolated mitochondria were analyzed. The levelof expression of the 30 kDa protein in the transfected MA-10 cells wasapproximately 7% of that observed in the Bt₂cAMP-stimulated MA-10 cells.Thus, these data indicate that the expression of the 30 kDa protein issufficient to induce steroid production in MA-10 cells in the absence ofhormone stimulation.

The present inventors have named this protein the &eroidogenic AcuteRegulatory protein, StAR. While not wishing to be bound by anyparticular theory regarding a mechanism of action, the following workingmodel for the acute regulation of steroidogenesis in Leydig cells byStAR is provided. The precursor protein is rapidly synthesized in thecytosol in response to hormone stimulation. The precursor binds to areceptor on the mitochondrial membrane and processing begins. Processingconsists of the N-terminus entering the mitochondrial matrix and beingcleaved to the 30 kDa form. It is during the processing that contactsites between the inner and outer mitochondrial membranes form and thisvery hydrophobic environment provides the medium through whichcholesterol may pass to the inner membrane. Thus, it may be theprocessing of StAR from a 37 kDa form to a 30 kDa form that isfunctionally active in the transport of cholesterol and results inincreased steroid production. This protein is also demonstrated to bindcholesterol (See Example 18).

EXAMPLE 4 Expression of StAR Precursor Protein in E. coli

The present example describes studies carried out to express the 37 kDaprecursor StAR protein in E. coli for overproduction thereof.

The E. coil expression vector, pKK233-2, (Clontech, Palo Alto, Calif.)contains an IPTG-inducible promoter (P_(trc)), a LacZ ribosome bindingsite, and a unique Nco1 cloning site that provides an ATG initiationcodon. Since expression of mitochondrial proteins tends to be toxic tobacterial host cells, basal levels of expression can be greatly reducedin E. coli strains that overproduce the lac repressor (lacIq) andoptimal expression can be achieved by induction for a short period oftime.

StAR cDNA was cloned in pSPORT vector (GIBCO Life Technologies,Gaithersburg, Md.) and this vector was used for PCR amplification of thecoding sequence for StAR. Primers were designed to introduce restrictionsites (Ncol at the 5′ end and HindMil at the 3′ end) for directionalsubeloning into the pKK233-2 vector. Ligation of the amplified StAR cDNAfragments with the pKK233-2 vector constructed a recombinantpKK233-2/StAR plasmid. The E. coli strains, JM109 and DH5αF′IQ, weretransformed with this plasmid and maintained in LB media (10 g tryptone,5 g yeast extract, 10 g NaCI, and 100 μg/ml ampicillin). For expression,a fresh culture (15-30 ml LB media) inoculated with the transformed E.coli was grown to an OD₆₀₀=0.6 at 37° C., then IPTG (isopropylP-D-thiogalactopyranoside) was added to a final concentration of 1 mM.Protein expression was induced at 30 ° C. for 2 hours and cells wereharvested. Cells were lysed by sonication in buffer containing 50 mMTris (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% NP-40, 0.5%sodium deoxycholate, 100 μg/ml phenylmethylsulfonyl fluoride, 2 pg/mlaprotinin, and 2 μg/ml leupeptin.

From staining patterns on gels, it is apparent that significant amountsof StAR protein were made. The extract of E. coli DH5aFIQ with the StARcDNA insert contained a 37 kDa polypeptide which was approximately 3% ofthe total cellular protein. This 37 kDa protein was not detected in E.coli without the plasmid. The protein reacted with the antibody raisedagainst amino acids 88-98 of StAR.

For isolation and purification of the StAR precursor protein, the soniclysate was ultracentrifliged (100,000×g, 1 h), and the supernatantpassed through an affinity column packed with Protein A agarose beadscrosslinked with the anti-StAR antibody having binding specificity foramino acids 88-98 of StAR. The fractions enriched for StAR precursorprotein were concentrated and further purified by passage through a gelfiltration column (packed with Sephadex G-75 beads). Flow-throughfractions were tested for the StAR precursor protein by Western blotanalysis and the purity was assayed using a silver staining method.

EXAMPLE 5 StAR is Hormonally Regulated and Developmentally Regulated

The present example provides studies that show that the production ofStAR protein is hormonally regulated, as well as developmentallyregulated in vivo.

MA-10 cells were stimulated with Bt₂cAMP and StAR MRNA levels weredetermined by Northern blot analysis. Within 1 hour of Bt₂cAMPstimulation, two major transcripts of approximately 3400nt and 160nt,and one minor transcript of 2700nt, were detected. StAR mRNAs weremarkedly induced (20×) after 2 hours of hormone stimulation with maximallevels obtained after 6 hours. Subsequent to the marked niRNA induction,the greatest induction (10 ×) in StAR protein was detected after 4 hoursof BtcAMP stimulation by immunoblot analysis. Comparatively lower levelsof StAR could be detected after 1 hour of hormone stimulation withmaximal levels accumulated within 8 hours.

Hormone-induced progesterone production rose above basal levels in MA-10cells typically within 1 hour with the greatest increase in rate ofsteroid output measured between 2-4 hours of hormone stimulation,consistent with the induction in StAR protein.

These data indicate that StAR is transcriptionally regulated by acAMP-mediated mechanism. Immunoblot analysis of several mouse tissuesindicates that StAR protein is expressed in the adrenal, ovary, andtestis, and is not expressed in brain, muscle, liver, kidney, spleen,heart, uterus, or placenta. The developmental expression of StAR wasassessed by in situ hybridization analysis of mouse embryonic tissue.Earliest detection of StAR transcripts was at embryonic day 10.5 (E10.5)in the genital ridge. By E12.5-E14.5, StAR was readily detected in theinterstitial cells of the testis and adrenal cortex and continued to beexpressed in the adult. StAR expression was absent in the ovary atE12.5-E14.5, but was abundant in the adult ovary. The developmentalpattern of expression for StAR parallels that observed previously forcytochrome P450scc which provides further supporting evidence for theimportance of StAR in steroid hormone biosynthesis.

The present inventors have generated stable transfected MA-10 cell linesthat constitutively synthesize the 3 0 kDa protein and that producesteroid constitutively at a level several fold higher (about 9x) thanbasal parental MA-10 cells.

EXAMPLE 6 Screening for Mutations in the StAR Gene for IdentifyingSteroid Hormone-Dependent Pathologies

The present example provides methods by which the nucleic acidmolecules, in particular, fragments of the nucleotide sequence of SEQ IDNO:1 or SEQ ID NO:15 may be used to detect mutations within the StARgene. These screening techniques may be used to identify a number ofdifferent pathologies; particularly steroid hormone-dependentpathologies, such as, for example, lipoid congenital adrenal hyperplasia(LCAH), adrenal hypoplasia congenita, hypogonadotropic hypogonadism,precocious puberty, or McCune-Albright syndrome.

The identification of highly conserved mutations and the development ofan appropriate screen would provide regulations and standards forclinical testing and screening for these metabolic disorders.

Lipoid Congenital Adrenal Hyperplasia (LCAH)

StAR DNA may be used for screening adrenal tissue obtained from patientswith Lipoid Congenital Adrenal Hyperplasia (LCAH) to test for a possiblerole of StAR in the disease state. This can be achieved with Southernblotting and hybridization with a cDNA probe. Fairly large DNArearrangements of greater than 500 bp may be detected in this manner.However, it may be that the mutations within the StAR gene resulting insteroid hornone-dependent pathologies are too small to detect bySouthern blotting. This would be the case if they are due to pointmutations or to small insertions, deletions or other rearrangements.

Smaller StAR gene mutations are detected by DNA sequencing which can beperforme4 on a genomic DNA template, a cDNA template prepared from RNAby reverse transcriptase, or on a PCR product. In an attempt to detectmutations rapidly, several methods are available; chemical cleavage,denaturing gradient electrophoresis (DGGE) and ribonuclease cleavage,and single strand conformation polymorphism. These methods and othersmay be used in conjunction with the present invention and may beperformed after PCR amplification of the DNA region under study.

Testicular tissue of two patients and genome DNA of a third patient withLCAH have mutations in the gene for StAR (Lin et al., Science,267:1828-1831, 1995) (incorporated herein by reference). These mutationsconsist of C to T transitions in the gene sequences, which resulted inthe premature insertion of stop codons. This resulted in the truncationof StAR protein by 28 amino acids in two of the patients and 93 aminoacids in another. These truncations were confnmed by Western analysisfollowing expression of the mutated cDNAs in COS cells. Virtually noneof the precursor form of StAR expressed from the cDNA of these patientswas converted to the mature mitochondrial form. Expression of the StARcDNA from these patients in COSl cells indicated that the proteinproduced was inactive in its ability to promote steroidogenesis, whereasthe normal protein resulted in an 8-fold increase in steroid productionwhen expressed.

EXAMPLE 7 Gene Therapy

This prophetic example describes some of the ways in which the presentinvention may be of use in the treatment of steroid hormone-dependentdisorders, especially those characterized as involving defects incholesterol transport.

A wild-type human StAR gene may be introduced into human tissue toprovide a wild-type copy of the gene and therefore, also a wild-typeprotein product, that may correct the genetic lesion that causes thesteroid hormone-dependent disorder.

Human adenovirus or retrovirus are means for introducing genes intotissue. Adenoviruses are double-stranded DNA tumor viruses with genomesizes of approximate 36 kb. As a model system for eukatyotic geneexpression, adenoviruses have been widely studied and wellcharacterized, and have already been used in a gene transfer system (seee.g., WO9506743, WO9502697, WO9500655, WO9428938, WO9419478, andWO9412649, each publication is incorporated by reference herein) . Thisgroup of viruses is easy to grow and manipulate, and they exhibit abroad host range in vitro and in vivo. In lytically infected cells,adenoviruses are capable of shutting off host protein synthesis,directing cellular machineries to synthesize large quantities of viralproteins, and producing copious amounts of virus.

In general, adenovirus gene transfer systems are based upon recombinant,engineered adenovirus which is rendered replication-incompetent bydeletion of a portion of its genome, such as E1, and yet still retainsits competency for infection. Relatively large foreign proteins can beexpressed when additional deletions are made in the adenovirus genome.For example, adenoviruses deleted in both E1 and E3 regions are capableof carrying up to 10 Kb of foreign DNA and can be grown to high titers.Persistent expression of transgenes follows adenoviral infection.

Particular advantages of an adenovirus system for delivering foreigngenes and their protein products to a cell include (i) the ability tosubstitute relatively large pieces of viral DNA with foreign DNA; (ii)the structural stability of recombinant adenoviruses; (iii) the safetyof adenoviral administration to humans; and (iv) lack of any knownassociation of adenoviral infection with cancer or malignancies; (v) theability to obtain high titers of the recombinant virus; and (vi) thehigh infectivity of adenovirus.

Further advantages of adenovirus vectors over retrovirses include thehigher levels of gene expression. Additionally, adenovirus replicationis independent of host gene replication, unlike retroviral sequences.Because adenovirus transforming genes in the E1 region can be readilydeleted and still provide efficient expression vectors, oncogenic riskfrom adenovirus vectors is thought to be negligible.

Patients testing positive for LCAH and for whom the medical indicationfor adenovirus-mediated gene transfer has been established, would betested for the presence of antibodies directed against adenovirus. Ifantibodies are present and the patient has a history of allergy toeither pharmacological or naturally occurring substances, application ofa test dose of on the order of 10³ to 10⁶ recombinant adenovirus underclose clinical observation would be indicated.

Recombinant adenovirus providing the wild-type StAR gene may be preparedand purified by any of a variety of methods, so as to provide apreparation suitable for administration to human subjects, including,but not limited to cesium chloride density gradient centriflgation, andsubsequently tested for efficacy and purity. Virus is administered topatients by means of intravenous administration in any pharmacologicallyacceptable solution, either as a bolus or as an infusion over a periodof time. Generally speaking, it is believed that the effective number offunctional virus particles to be administered would range from 5×10¹⁰ to5×10¹².

Patients would remain hospitalized for at least 48 hr to monitor acuteand delayed adverse reactions. Serum levels of a protein product may bemonitored or Southern blots may be performed to follow the efficacy ofthe gene transfer. Adjustments to the treatment may include adenovirusconstructs that use different promoters or a change in the number of pfuinjected.

EXAMPLE 8 Expression of StAR induces Steroid Production in the absenceof Hormone Stimulation

The present example demonstrates that StAR expression induces steroidproduction in the absence of hormone stimulation.

Transient transfection of COS 1 cells with StAR resulted in a severalfold increase in steroid production in the absence of hormonestimulation. Transient transfection of COS 1 cells with StAR wasobserved to provide in a several fold increase in steroid production.Also, increases in StAR MRNA and protein closely paralleled steroidproduction indicating a temporal relationship in these parameters.

EXAMPLE 9 Calcium Stimulates StAR Protein

The effect of changes in [Ca²⁺]c on intramitochondrial cholesterol andthe distribution of StAR protein in mitochondria during activation byCa²+ is described in the present example.

In adrenal glomerulosa cells, angiotensin II (Ang II) stimulatesaldosterone synthesis through rises of cytosolic calcium, [Ca²⁺]. Therate-limiting step is the transfer of cholesterol to the innermitochondrial membrane, where it is converted to pregnenolone by theP450 side chain cleavage enzyme (P⁴⁵⁰scc). This transfer is believed tooccur as the 37 kDa precursor of the Steroidogenic Acute Regulatory(StAR) protein is imported into the mitochondria.

Freshly-prepared bovine zona glomerulosa cells were stimulated with Ang1 (10 nM) or submitted to a cytosolic Ca²⁺ clamp (600 nM) for 2 h.Mitochondria were isolated and subionated into outer membranes (OM),inner membranes (IM) and contact sites (CS). Cholesterol content wasdetermined by the cholesterol oxidase assay.

When glomerulosa cells were exposed to Ang II, a marked increase ofcholesterol in CS occurred (to 172±28% of controls, n=3). No significantchanges were detected in OM cholesterol, suggesting a stimulation ofcholesterol supply to the mitochondria in response to Ang II.Stimulation of intact cells with Ca?+led to a marked decrease incholesterol content of OM (to 54±24% of controls, n=5). Cycloheximidespecifically and significantly reduced Ca²⁺-activated cholesteroltransfer to IM. Western blot analysis revealed a cycloheximide-sensitiveincrease of StAR protein (to 141±14% of controls, n=5) in mitochondrialextracts of Ca²⁺-mobilizing agents, newly synthesized StAR accumulatesin IM after transiting through CS. One of the main functions of the Ca²⁺messenger is to increase cholesterol supply to the P450_(scc) enzyme byenhancing endogenous intermembrane cholesterol transfer. The import ofStAR protein to IM is accompanied by cholesterol transfer, thuspromoting the activation of the steroidogenic cascade.

EXAMPLE 10 Angiotensin II Stimulates Intramitochondrial CholesterolTransfer and StAR Protein in Bovine Adrenal Glomerulosa Cells

The effect of Ang II on intramitochondrial cholesterol and thedistribution of StAR protein in submitochondrial fractions duringactivation by Ang II is described in the present example.

Freshly-prepared bovine zona glomerulosa cells were stimulated with AngII (10 nM) or submitted to a cytosolic Ca²+clamp (600 nM) for 2 h, asdescribed in Example 9. Mitochondria were isolated and subfractionatedinto outer membranes (OM), inner membranes (IM) and contact sites (CS).Cholesterol content was determined by the cholesterol oxidase assay,also as described in Example 9.

StAR protein in Ang II stimulated (to 157% of controls, n=2) glomerulosacells. Ang II increased StAR in IM, and this effect was prevented bycycloheximide.

EXAMPLE 11 StAR and a Water Soluble CaM Kinase IH Inhibitor

The present example demonstrates the effect of a water soluble CaMkinase II inhibitor on the agonist inductor of StAR protein. The watersoluble CaM kinase II inhibitor, KN93, was used in the present exampleand is representative of the water soluble CaM kinase II inhibitorsgenerally.

The human adrenocortical carcinoma-derived cell line (H295R) was used.This cell line secretes multiple steroids, including aldosterone andcortisol. This cell line is an appropriate model system to investigatethe acute regulation of human aldosterone synthesis, as described inBird et al (1993) (Endocrinology, 133, 1555-1561), which is specificallyincorporated herein by reference.

To further investigate the site of KN93 action, the effect of KN93 onagonists induction of the StAR protein, shown herein to regulatemovement of cholesterol from the outer to the inner mitochondrialmembranes, was examined. The amount of StAR protein was increasedfollowing treatment of H295R cells with angiotensin II (Ang II)potassium (K), and Bay K (a calcium channel activator) as shown by thepresent investigation (See Clark et al., 1995, Mol. Cell Endocrinol.,115:215-19). KN93 at concentration between 1 and 3 μM, which blockedsteroidogenesis by 60 to 80%, did not affect induction of StAR proteinby Ang II, K⁺, or Bay K. These results support the finding that CaMkinase II is involved in the process of cholesterol mobilization to themitochondria.

Materials and Methods

Materials

[Val⁵]-Angiotensin II acetate (Ang II), potassium chloride,22R-hydroxlycholesterol (22ROHChol), d-aldosterone, hydrocortisone(cortisol) and laboratory reagents were from Sigma Chemicals (St. Louis,Mo.). Dibutyryl cAMP (dbcAMP) was from Aldrich Chemicals (Milwaukee,Wisc.). (=)-Bay K 8644 (Bay K) was from Research BiochemicalsInternational (atick, MA). The protein kinase inhibitor KN93(2-[-(2-hydroxyethyl)-N-4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine)was from Seikagaku America, Inc. (Ijamsville, Md.). The calmodulininhibitor compound R 24571 (calmidazolium) was from JanssenPharnaceutica (Berse, Belgium).

Cell culture

H295R cells were initially obtained as NCI-H295 cells from the AmericanType Culture Collection (Rockville, Md.), and then selected as describedpreviously (Bud et al (1993)). Reflecting growth and culture differencesbetween the original ATCC cells and the selected subpopulation, thesecells are designated as H295R cells and are available from ATCC as such.Cells were maintained in a 1:1 mixture of Dulbecco's modified Eagle'sand Ham's F 12 (DME/F12) medium containing pyridoxine HCI, L-glutamineand 15 mM Hepes (Gibco BRL; Gaithersberg, MD); medium was supplementedwith insulin (6.25 μg/ml), transferrin (6.25 μg/ml), selenium (6.25ng/ml), bovine serum albumin (1.25 mg/ml), and linoleic acid (5.35μg/ml) added in the form of 1% ITS plus (Collaborative BiomedicalProducts; Bedford, Mass.). In addition, cells were grown in the presenceof either 2% low protein serum replacement (Sigma Chemicals; St. Louis,Mo.) or 2.5% Nu serum type I (Collaborative Biomedical Products;Bedford, MA), as well as antibiotics. Cells were maintained and grown in75 cm² flasks at 37° C. under an atmosphere of 5% CO₂/95% air,subcultured onto 12 well plates and used for experiments 48 hours lateras indicated below.

Stimulation ofsteroid secretion and analysis ofsteroids

Subcultured cells were maintained 24 hours in DME/F12 medium containing0.2% calf serum, 0.01% BSA and antibiotics (low serum). Cells werepreincubated with KN93 or calmidazolium for 30 minutes at 37° C. Freshlow serum medium containing the agents as indicated was then added tothe cells and the incubation carried out at 37° C. for the indicatedtimes.

The aldosterone and cortisol contents of medium recovered from each wellwere determined with aldosterone and cortisol standards prepared inlow-serum medium by commercial aldosterone and cortisolradioimmunoassays (Diagnostic System Laboratories; Webster, Tex.).Results of aldosterone and cortisol assays were normalized to cellularprotein per well, expressed as pmol per mg cell protein and transformedto percentage of the control response where indicated. IC₅₀ values forcalmidazolium and KN93 were calculated by taking the difference betweenbasal and stimulated values as 100%.

Protein determination

Cells were solubilized in Tris-HCI (50 mM pH 7.4) containing NaCl (150mM), SDS (1%), EGTA (5 mM), MgCl (0.5 mM), MnCl (0.5 mM), andphenylmethylsulfonylfluoride (PMSF 0.2 mM), and stored frozen at -20° C.Protein content of samples was then determined by the bicinchoninic acidprotein assay, using the BCA assay kit (Pierce, Rockford, Ill.).

Immunoblot analysis

For each treatment, cells were solubilized as described above and anequivalent amount of protein (30 μg) for each sample were separated bySDS-PAGE (12.5%) and then electrophoretically transferred topolyvinylidene difluoride membrane as previously described (Clark et al(1994) J. Biol. Chem., 269:28314-322, reference specificallyincorporated herein by reference). Immunoblot analysis was as described(Id) using a rabbit sera containg the specific StAR antipeptide antibody(see ANTIBODIES infra) as the primary label and a donkey anti-rabbit IgGconjugated with horseradish peroxidase (Amersham, Arlington Heights,Ill.). The specific signal was detected by chemiluminescence assay usingthe Renaissance kit from DuPont NEN (Boston, Mass.) and the StARspecific bands were quantitated by Biolmage Visage 2000computer-assisted image analysis (BioImage, Ann Arbor, Mich.). Differentexposure times were used to insure linearity.

Statistical analysis

Statistical analysis of the data was by analysis of variance, followedby Student-Newman-Keuls multiple comparison analysis or, for transformeddata, by the Mann-Whitney U test.

KV93 effects on StAR induction

The data above suggest that CaM kinase II plays a role insteroidogenesis prior to P450scc conversion of cholesterol topregnenolone. StAR protein levels were elevated by Ang II, K⁺, and Bay Kin H295R cells. To discriminate between the potential sites of CaMkinase II involvement in aldosterone production, the effects of KN93 onStAR protein expression were also examined. Agonist-induction of StARprotein expression was not affected by co-treatment with KN93 at aconcentration (3 μM) which potently inhibited aldosterone production.

EXAMPLE 12 Transcription Repressor DAX-1 Inhibits StAR mRNA and ProteinSynthesis; Role of StAR in Screening for DSS

The overexpression of DAX-1 in Y-1 adrenal tumor cells results in acomplete inhibition of steroid synthesis and an accompanying inhibitionof StAR MRNA and protein synthesis. The present example demonstratesthis phenomenon.

Male to female sex reversal has been observed in individuals withduplications of the short arm of the X chromosome. The study of Xpduplicated patients demonstrated that sex reversal results from thepresence of two active copies of the DSS (Dosage Sensitive Sex Reversal)locus. A double dosage of the DSS disrupts testis formation while itsabsence is compatible with a male phenotype, suggesting a role for DSSin ovarian development and as a link between ovary and testis formation.DSS has been mapped to a 160bp region of human chromosome Xp21, whichincludes the adrenal hypoplasia congenita (AHC) locus.

It is contemplated by the present inventors that the DSS is DAX-1, anunusual member of the nuclear hormone receptor superfamily. DAX-1 mapsto the DSS critical region, and is responsible for X-linked adrenalhypoplasia congenita (See Swain et al. 1996, Nature Genetics, 12:404-409).

The present inventors contemplate that when a double dose of DSS ispresent, it can bind to SF1 sites at a higher amount than usual, thusturning off SF1 gene (such as StAR, p450 and 3-bHSD). One would expectto see no testosterone synthesized, and the male sex organs would notdevelop. Hence, the present observation that DAX-1 inhibits StARsynthesis suggests a use of StAR nucleic acid in a screening assay forthe disease DSS and constitutes a method of use as part of the presentinvention.

SCREENING FOR DOSAGE SENSIIIE SEX REVERSAL

The present example describes the utility of using mouse StAR MnRNAsequence, or the StAR MRNA sequence as defined in SEQ ID NO: 14, whichis the coding region of the mouse mRNA sequence from nucleotide position210 to 931, in a screening assay for DSS. This region shares about 84%identity with the human StAR MRNA sequence from nucleotide position 267to 988 (SEQ ID NO: 15), in a method for screening a patient sample forDosage Sensitive Sex Reversal (DSS). The screening procedure may also beconducted using the nucleic acid sequence, SEQ ID NO: 15, or a fragmentof that sequence. It is anticipated that any of the primers describedherein may be used as a PCR primer to generate an about 400 bp piece ofnucleic acid that may be used for the present screening assay.

A tissue sample from a patient to be screened would be tested todetermine the presence of nucleic acid that demonstrated hybridizationto the probe sequence for StAR by RT-PCR using any one of the abovedescribed StAR encoding sequences. If presence of StAR nucleic acid isdetected, the patient samle will be determined not to evidence DSS. Ifpresence of StAR nucleic acid is detected, then the patient will bedetermined to have DSS, or to be at risk of developing this disease.This screening protocol may be used together with or clinicallydetectable symptoms of the patient in determining the appropriatetreatment for the patient.

Children diagnosed with DSS bave been fouid to have elevated, and mosttimes twice, the level of a mutated form of the protein, DAX-1 (SeeZanaria et a. (1994) Nature 372(15):635-641). DSS is a disease thataffects primarily males, and that causes afflicted males tophenotypically revert to females. (See Namabe et al. (1992) Hum. Genet.,90:211-214). The present invention contemplates the use of StAR in thediagnosis of this disease, as it is contemplated that the StAR gene isnot functional or is not expressed at sufficient levels in thesepatients, thus resulting in the phenotypically detectable sex reversal.

EXAMPLE 13 Mouse StAR Transcripts and Human StAR Transcripts Specificfor StAR MRNA

Three transcripts specific for StAR MRNA have been detected in the mouse(˜1.6 kb, 2.7 kb, and −3.4 kb) and in the human (˜1.6 kb, 4.4 kb, and˜7.5 kb).

Differences in the lengths between the mouse and human trrnscripts wereidentified. The difference in length is attributed, at least in part, toa difference in the length of the 3′-intranslated regions.

The additional length was determined to exist at a site 31 of the Xholcleavage site, as determined in the following study:

Digest λ DNA purified from bacteriophage with Not II/Sal IIXho I torelease the Not I and Sal I insert (cloning strategy) and Xho I todetermine which end of the cDNA has the additional sequences.

Results: StAR cDNA

Clone 1 (StAR CDNA)—1500 bp, mouse StAR sequence expect—61 bp and 939 bpfragments

Clone 2 (FP#1)—1500 bp

Clone 3 (FP#12)—1650 bp

Clone 4 (FP#5)—2400 bp

Clone 5 (FP#7)—2400 bp

Clone 6 (FP#16)—2350 bp

Clone 7 (FP#13)—2800 bp

Conclusion: additional length in human StAR cDNA is 3′ of the Xho Isite.

EXAMPLE 14 Mouse StAR Identity to Other Species

Full length cDNA clones for StAR have been isolated for the mouse andhuman (FIG. 3). cDNA for human StAR was isolated from a human adrenallibrary. The deduced amino acid sequence from the human StAR DNA wasabout 87% identical to the mouse sequence. The cDNA's have approximately84% homology. The structural gene for StAR has also been isolated andcharacterized for both the mouse and human. The genes span 6.5 kb in themouse and 8 kb in the human with the intronic sequences contributing toincreased length in the human. Both are organized into seven exons andsix introns with exons III-XI being of identical size.

A StAR pseudogene was identified by RT-PCR amplification of RNA fromhuman testis and PCR amplification of human genomic DNA (Sugawara et al(1995), Lin et al. (1995)). Sequence analysis of the pseudogeneindicated that it lacks introns and has several nucleotide insertions,deletions, and substitutions (Sugawara et al. (1995)). However,detection by RT-PCR suggests that the pseudogene is transcribed.

Southern blotting of somatic cell hybrids followed by fluorescent insitu hybridization was used to map the human structural gene tochromosome 8p. 11.2 and the pseudogene to chromosome 13 (Sugawara et al(1995)). The human promoter, a 1.3-kb upstream sequence, can confer bothbasal and cAMP-dependent transcriptional activation of a luciferasereporter gene in Y1 mouse adrenal cells (Id). Similarly, 1 kb of the5′-upstream sequence of the mouse StAR gene has been observed to driveexpression of human GH reporter in Y1 cells. The suggestion that StAR isa potential target for SF-1 is supported in studies that demonstratethat SF-1 binds to both the murine and human promoters and contributesto the transcriptional activity of STAR.

StAR protein sequence is highly conserved with 85%-88% identity andgreater than 90% similarity in the species studied to date (FIG. 5). Inaddition, a partial cDNA has been isolated from the sheep that has 80%identity with the corresponding regions of the other species. Thegreatest divergence appears to center around the putative mitochondrialsignal sequence cleavage site described here for the mouse sequence.This region of the protein contains an amino acid motif that is highlyconserved in presequences that undergo a sequential two-step cleavage bythe matrix-processing protease and the mitochondrialintenmediate-processing peptide, respectively.

The submitochondrial localization of StAR has been determined usingprotein-A gold labeling of immuno-reacted StAR in mouse adrenal zonafasciculata cells. Colloidal gold particles were concentrated within themitochondria to the intermembrane space and the intermembrane space sideof the cristae membrane (King et al (1995)).

In vitro transcription/translation systems have been used to demonstratethat isolated mitochondria are competent to import and process bothmouse and bovine StAR protein. Rat heart mitochondria were used for theimport assay with bovine StAR, indicating import of StAR is notdependent upon factors specific to mitochondria isolated fromsteroidogenic tissues. The ability of StAR to increase steroidproduction has also been confirmed in an in vitro reconstituted system.StAR protein added to mitochondria isolated from MA-10 mouse Leydigtumor cells has been observed to result in a time- and dose-dependentincrease in pregnenolone synthesis. This stimulation was shown to bespecific for StAR in that pregnenolone synthesis was not affected byaddition of another mitochondrial imported protein, adrenodoxin.

Computer analysis of the StAR protein sequence has identified threeputative PKA/Cam kinase II phosphorylation sites and one PKCphosphorylation site (FIG. 5). Site-directed mutagenesis of StAR cDNA,which changes these putative target residues, will be used to determineif the phosphorylation of StAR itself is required for mitochondrialimport, and that phosphorylation of StAR is directly linked to thesteroidogenic response of the cell to hormone stimulation.

The high degree of similarity between the nucleotide sequence for themouse and human StAR does not extend into the 5′-flanking regions of thegenes. However, the human StAR promoter also contains a putativeSF-l/AdBP4 binding site located −926/−918 relative to the start site oftranscription in addition to two putative Spl consensus consequences(Sugawara et al (1995)).

The coding region of the human StAR cDNA has been characterized and isshown in SEQ ID NO: 19. The coding nucleotides are capitalized, whereasthe untranslated nucleotides are in lowercase.

Toward more definitive studies on the mechanisms regulating StAR geneexpression, approximately 1 kb and 1.3 kb of the 5′-flanking regions ofthe mouse and human gene, respectively, have been isolated andsequenced. Inspection of the murine StAR promoter does not provide manyclues to putative cis-acting regulatory elements; it lacks a canonicalTATA box and does not contain a consensus cAMP-responsive element. Thus,like most of the cytochrome P450 steroid hydroxylase genes that are alsoregulated by cAMP but lack classical CREs, the regulatory regions ofStAR may be unique.

EXAMPLE 15 StAR is Regulated by Oestradiol

This present study demonstrates that oestdiol alters components of thesteroidogenic pathway between cholesterol and P450 scc.

Oestradiol inhibits testosterone (T) secretion in rams, and at leastpart of this action is gonadotrophin-independent. There is therefore alocal effect on testicular steroidogenesis. Low-titre immunization oframs against oestradiol increases T secretion without affectingtesticular steroidogenic enzyme activity or expression. Adult rams,during the breeding and nonbreeding seasons, were injected (iv every 3-4days) with enough oestradiol antiserum to maintain an antibody titre ofabout 1:200. At this titre, the antibody does not cross-react with T.Blood samples were taken every 20 minutes for 10 h (d 21 ofimmunization) for pulsatile T and LH measurements. Pooled samples wereused for assay of cholesterol, HDL and LDL+VLDL concentrations. Thetestes were then removed and RNA extracted for measurement of LDLreceptor and StAR MRNA levels.

Mean, basal plasma T concentrations were not altered by treatment or byseason (P>0.05). Relative levels of StAR MRNA were 67% higher inbreeding vs nonbreeding season (P<0.01), and 21% higher in immune vscontrol rams (P<0.05) irrespective of season. Low-titre oestadiolimmunization increases T secretion by increasing StAR mRNA abundance andthus the delivery of cholesterol to P450scc. These data provide evidencethat StAR is regulated by oestradiol in the testis.

EXAMPLE 16 Binding of StAR Protein to a Mitochondrial Membrane ProteinComplex

The following studies demonstrates that cholesterol transfers to theinner mitochondrial membrane only as StAR protein is being imported.

A protein complex was purified from MA-I 0 cell mitochondrial membranesusing an affinity column constructed by crosslinking the C-teiminus ofcommercially synthesized StAR signal peptide to Sepharose 4B beadsmodified with hydrazine. Specific binding of StAR signal peptide to thecomplex was examined. When biotinylated StAR signal peptide wasincubated with the protein complex, followed by crosslinking withdisuccinimidyl suberate, it shifted in size from 3.5 kDa toapproximately 300 kDa upon one dimensional SDS polyacrylamide gelelectrophoresis. The binding of biotinylated StAR signal peptide to theprotein complex was completely inhibited by preincubation with 200 μMunlabeled StAR signal peptide. Conversely, preincubation with 200 μM ofthe signal peptide of ornithine transcarbamylase (OTC) or 100 PM of thesignal peptide of the F1-ATPase-B subunit, two other mitochondrialproteins, did not show similar inhibitory effects on binding. Thisprotein complex was then incorporated into liposomes for furtherexamination. Incubation of ³⁵S-labeled STAR signal peptide with theliposomes resulted in a sharp peak of radioactivity which co-eluted withthe liposome fiaction recovered from a Sephacryl S-200 column.Preincubation of unlabeled StAR signal peptide with the liposomesreduced total radioactivity by 65% in this fraction, whereas OTC signalpeptide did not reduce the binding to the liposomes. These resultssuggest that the signal peptide region of the StAR protein is capable ofspecifically binding to a mitochondrial membrane protein complex.

EXAMPLE 17 Corticotropin-Releasing Hormone (CRE) andTestosterone—Effects of StAR

CRH treatment of MA-10 mouse Leydig tumor cells results in a dosedependent stimulation of testosterone production (Biol Reprod 53:620-626(1995)). In view of this observation, the effects of CRH on thesynthesis of the StAR protein in these cells was examined.

Treatment of MA-10 cells with CRH resulted in a dose-dependent increasein the synthesis of the StAR protein with a maximal response observed at1 μM. The maximal response to 1 μM CRH was seen at 4 hr followingstimulation. Treatment with the cAMP analog, dbcAMP, also resulted in adose-dependent increase in both StAR and steroid synthesis, reaching amaximum at 1 μM. The maximum response to dbcAMP occurred at 6 hr poststimulation. While hCG treatment of MA-10 cells also resulted in anincrease in StAR synthesis, the levels obtained were significantly lowerthan those seen with CRH or dbcAMP. CRH treatment in the presence of hCGresulted in a higher level of StAR synthesis than that seen with maximaldoses of CRH or hCG alone. These results indicate that CRH, like hCG anddbcAMP, can stimulate the synthesis of testosterone in MA-10 Leydigtumor cells and apparently does so by increasing the synthesis of theStAR protein.

EXAMPLE 18 StAR as a Cholesterol Binding Protein

The present study demonstrates the cholesterol binding capacity of StARand its potential utility for binding and hence use in the regulationand/or reduction of cholesterol.

For these studies StAR proteins were obtained from three sources; (1)MA-10 cells stimulated with dbAMP; (2) monkey kidney cells (COS 1)transfected with StAR cDNA; and (3) expression of a recombinantpKK233-2/StAR plasmid in E. coli . Filtration radioassays andSteady-State (native) polyacrylamide gel electrophoresis were used todetect cholesterol binding to StAR. Results from these radioassaysdemonstrated low levels of specific binding (2040% of the total binding)of cholesterol to StAR. Fluorescent sterol binding assay results supportthe finding that the binding of NBD-cholesterol to StAR occurs with anaffinity in the nanomolar range. Thus, this study has provided evidencethat cholesterol can bind to the StAR protein with high affinity.

EXAMPLE 19 StAR Binding Site for SF-1 AdBP4

In the mouse, a sequence motif that matches the known requirements forbinding the orphan nuclear receptor, SF-l/AdBP4, is located at position−128/−135 relative to the transcriptional start site. SF-1/AdBP4 bindingsites are present in the promoters of all the steroid hydroxylase genes,and SF-1/AdBP4 has been shown to transcriptionally regulate theirexpression in a cAMP-dependent manner. Thus, StAR may represent anothertarget for SF- IAdBP4.

All of the compositions and methods disclosed and claimed herein can bemade and executed ithout undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

Alberta et al., (1989) J. Biol. Chem. 264,2368-2372.

Almahbobi, et al., (1992) Exp. Cell Res. 200,361-369.

Andersson, et al., (1989) J. Biol. Chem 264, 8222-8229.

Ardail, et al., (I 991) J. Biol. Chem. 266, 7978-7981.

Ascoli, M. (1981) Endocrinology 108, 88-95.

Bonner and Laskey, (1974) Eur. J Biochem. 46, 83-88.

Bradford, M. (1976) Anal. Biochem. 72, 248-254.

Brown, et al., (1992) Mol. Cell. Endrocrinol. 83, 1-9.

Camacho, et al., J Clin. Endocrinol. Metab. 28:153-161, 1968.

Caracciolo et al. (1989) Science, 245:1107.

Chanderbhan, et al., (1982) J Biol. Chem. 257, 8928-8934.

Chomczynski and Sacchi, (1987) Anal. Biochem. 162, 156-159.

Clark and Waterman, (1991) J. Biol. Chem. 266, 5898-5904.

Cooke, et al., (1975) Biochem.J 150, 413418.

Crivello and Jefcoate, (1980) J. Biol. Chem. 255, 8144-8151.

Davis and Garren, (1968) J. Biol. Chem. 243, 5153-5157.

Degenhart, et al., Acta Endocrinologia 71:512-518, 1972.

Deutscher, (1990) Methods in Enzymology, 182, Guide to ProteinPurification, Academic Press, Inc.

Elliott, et al, (1993) Endocrinology 133, 1669-1677.

Epstein and Orre-Johnson, (1991) J Biol. Chef 266, 19739-19745.

Ferguson, (1963) J. BioL Chem. 238, 2754-2759.

Freeman, (1987) J. BioL Chem. 262,13061-13068.

Gamier, et al., (1993) Endocrinology 132, 444-458.

Garren, et al., (1 965) Biochemistry 53, 1443-1450.

Glick, et al., (1991) Trends Cell Biol 1, 99-103.

Green and Orme-Johnson, (1991) J. Steroid Biochem. Molec. Biol.40,421429.

Hall and Almahbobi, (1992) J. Steroid Biochem. Molec. Biol. 43, 769-777.

Hartl, F-U. (1986) Cell 47,,939-951.

Hauffa, et al., Clin. Endocrinol. 23:481-493, 1985.

Hawley-Nelson, et al., (1993) Focus 15,73-79.

Haynes, etal., (1959) J. Biol. Chem. 234, 1421-1423.

Hendrick, et al., (1989) Proc. Natl. Acad Sci. US.A., 86,4056-4060.

Jefcoate, et al., (1987) J. Steroid Biochem. 27, 721-729.

Jefcoate, et al., (1986) Endocr. Res. 12, 315-350.

Jefcoate, et al., (1992) J. Steroid Biochem. Molec. Biol 43, 751-767.

Jefcoate, et al., (1974) Eur. J Biochem. 42, 539-551.

Kalousek, et al. (1988) Proc. Natl. Acad Sci USA. 85, 7536-7540.

Karaboyas and Koritz, (1965) Biochemistiy 4, 462-468.

Kiebler, et al., (1993) J. Memb. Biol 135, 191-207.

Koizumi etal., Clin. Chim. Acta 77:301-306, 1977.

Krueger and Orme-Johnson, (1983) J. Biol. Chem. 258, 10159-10167.

Laemmli, U.K. (1970) Nature 227, 680-688.

Lin et al., J. Clin. Invest. 88:1955-1962, 1991

Mendelson, et al., (1975) Biochim. Biophys. Acta 411, 222-230.

McEnery, et al., (1992) Proc. Natl. Acad Sci. U.S.A., 89, 3170-3174.

Menrifield, R., J. Am. Chem. Soc., 85:2149, 1963.

Mertz and Pedersen, (1989) Endocr. Res. 15, 101-115.

Farrell, P.H. (1975) J Biol. Chem. 250, 4007-4021.

Pedersen and Brownie, (1983) Proc. Natl. Acad. Sci USA. 80, 1882-1886.

Pedersen and Brownie, (1987) Science 236, 188-190.

Pon et al., (1986) J Biol. Chem. 261, 13309-13316.

Pon et al., (I1986) Endocr. Res. 12, 429-446.

Privalle, et al., (1983) Proc. Natl. Acad Sci. USA. 80, 702-706.

Python, et al., (1993) Endocrinology 132, 1489-1496.

Reddy, et al., (1993) BioTechniques 15,444-448.

Remington: The Science and Practice of Pharmacy, 19th edition, Volumes Iand 2, A. R. Gennaro, ed., Mack Publishing Co. Easton, Pa., 1995.

Resko, et al. (1974) Endocrinology 94, 128-135.

Sambrook, et al., (1989) Molecular Cloning, A Laboratory Manual, 2ndEd., Cold Springs Harbor Laboratory, Cold Springs Harbor, N.Y.

Saiki, et al., (1988) Science 239, 487-494.

Sala, et al., (1979) J Biol. Chem. 254, 3861-3865.

Sanger, et al., (1977) Proc. Nati. Acad Sci. US.A. 74,5463-5467.

Schleyer and Neupert, (1985) Cell 43,339-350.

Schwaiger, et al., (1987) J Cell Biol. 105,235-246.

Simbeni, et al., (1990) J. Biol. Chem. 265, 281-285.

Simbeni, et al., (1991) J. Biol. Chem. 266, 10047-10049.

Simpson, et al., (1972) Eur. J Biochem. 28, 442-450.

Stocco and Kilgore, (1988) Biochem. J 249,95-103.

Stocco and Chaudhary, (1990) Cell. Signal. 2, 161-170.

Stocco and Chen, (1991) Endocrinology 128, 1918-1926.

Stocco and Sodeman, (1991) J Biol. Chem. 266, 19731-19738.

Stocco, D. M. (1992) J. Steroid Biochem. Molec. Biol. 43, 319-333.

Stocco and Ascoli, (1993) Endocrinology 132, 959-967.

Stocco, et al, (1993) Endocrinology 133, 2827-2832.

Stocco and Clark, (1993) J. Steroid Biochem. Mole. Biol. 46, 337-347.

Stone and Hechter, (1954) Arch. Biochem. Biophysics 51, 457-469.

Towbin, et al., (1979) Proc. Natl. Acad. Sci. USA. 76, 4350-4354.

van Amerongen, et al., (1989) Biochim. Biophys. Acta 1004, 36-43.

von Heijne, G. (1986) EMBO J 5, 1335-1342.

von Heijne, et al., (1989) Eur. J Biochem. 180, 535-545.

Guo, et al. (1995) JAMA 274, 324-330.

Yanase, et al. (1996) J. Clin. Endocrinol. Metab. 81, 530-535

Muscatelli, et al. (1994) Nature 372, 672-676

Zanaria, etal. (1994) Nature 372, 635-641

Swain, et al. (1996) Nature Genetics 12, 404-409

19 1466 base pairs nucleic acid single linear other nucleic acid /desc =“DNA” unknown 1 GTCGACCCAC GCGTCCGCTC AGGACCTTGA AAGGCTCAGG AAGAACAACCCTTGAGCACC 60 TCAGCACTCA GCATGTTCCT CGCTACGTTC AAGCTGTGTG CTGGAAGCTCCTATAGACAT 120 ATGCGGAATA TGAAAGGATT AAGGCACCAA GCTGTGCTGG CCATTGGCCAAGAGCTCAAC 180 TGGAGAGCAC TGGGGGATTC CAGTCCCGGG TGGATGGGTC AAGTTCGACGTCGGAGCTCT 240 CTGCTTGGTT CTCAACTGGA AGCAACACTC TATAGTGACC AGGAGCTGTCCTACATCCAG 300 CAGGGAGAGG TGGCTATGCA GAAGGCCTTG GGCATACTCA ACAACCAGGAAGGCTGGAAG 360 AAGGAAAGCC AGCAGGAGAA CGGGGACGAA GTGCTAAGTA AGATGGTGCCAGATGTGGGC 420 AAGGTGTTTC GCTTGGAGGT GGTGGTAGAC CAGCCCATGG ACAGACTCTATGAAGAACTT 480 GTGGACCGCA TGGAGGCCAT GGGAGAGTGG AACCCAAATG TCAAGGAGATCAAGGTCCTG 540 CAGAGGATTG GAAAAGACAC GGTCATCACT CATGAGCTGG CTGCGGCGGCAGCAGGCAAC 600 CTGGTGGGGC CTCGAGACTT CGTGAGCGTG CGCTGTACCA AGCGCAGAGGTTCCACCTGT 660 GTGCTGGCAG GCATGGCCAC ACATTTTGGG GAGATGCCGG AGCAGAGTGGTGTCATCAGA 720 GCTGAACACG GCCCCACCTG CATGGTGCTT CATCCACTGG CTGGAAGTCCCTCCAAGACT 780 AAACTCACTT GGCTGCTCAG TATTGACCTG AAGGGGTGGC TGCCGAAGACAATCATCAAC 840 CAGGTCCTAT CGCAGACCCA GATAGAGTTC GCCAACCACC TGCGCAAGCGCCTGGAAGCC 900 AGCCCTGCCT CTGAGGCCCA GTGTTAAGGA CTGTCCACCA CATTGACCTGCAAATCATTG 960 GAAGCTCTCA CAGGAAGCCT GCAAGTCTGT CCATCTTCAG CTAACAGCATCGGGAGGGGT 1020 GGTAGTCAGG AGACACTAGG ACTGACTGGT AAAATCAGGA TCAGCAAAATAGAAATGAGG 1080 CTTAGAATAA AAGTTCTCTA GTGTCTCCCA CTGCATAGCT GTGAAGGCTAAGGGATAAGT 1140 AGCTATGAAA CCTTTCATCT AGGCTTGTAT ATGCTGACCT AAAAGACACCAGCAGCTACG 1200 AACAGGGGAT GCTAAGGATC GGGAACTGTT GTCTTACCAG CTCCAAATGTCACTACCTGA 1260 AGGCAGTGTG CACACAAAGC AAGGTCTTGC CTAGGAAACT CTGTAAAAGTTCTCCTCTGT 1320 AAAAGGCCAG AACTTGAATG AAACTACCTA CAAAGGGCCT TTCCAGAGTATTCCAACTTT 1380 TCTCTGAGGA GAAATGAAAC CATCATTGTG CCGACTTCCC TACTAATCCCATGACAATAA 1440 AGAACATACA TAAAAAAAAA AAAAAA 1466 276 amino acids aminoacid single linear protein unknown 2 Met Phe Leu Ala Thr Phe Lys Leu CysAla Gly Ser Ser Tyr Arg His 1 5 10 15 Met Arg Asn Met Lys Gly Leu ArgHis Gln Ala Val Leu Ala Ile Gly 20 25 30 Gln Glu Leu Asn Trp Arg Ala LeuGly Asp Ser Ser Pro Gly Trp Met 35 40 45 Gly Gln Val Arg Arg Arg Ser SerLeu Leu Gly Ser Gln Leu Glu Ala 50 55 60 Thr Leu Tyr Ser Asp Gln Glu LeuSer Tyr Ile Gln Gln Gly Glu Val 65 70 75 80 Ala Met Gln Lys Ala Leu GlyIle Leu Asn Asn Gln Glu Gly Trp Lys 85 90 95 Lys Glu Ser Gln Gln Glu AsnGly Asp Glu Val Leu Ser Lys Met Val 100 105 110 Pro Asp Val Gly Lys ValPhe Arg Leu Glu Val Val Val Asp Gln Pro 115 120 125 Met Asp Arg Leu TyrGlu Glu Leu Val Asp Arg Met Glu Ala Met Gly 130 135 140 Glu Trp Asn ProAsn Val Lys Glu Ile Lys Val Leu Gln Arg Ile Gly 145 150 155 160 Lys AspThr Val Ile Thr His Glu Leu Ala Ala Ala Ala Ala Gly Asn 165 170 175 LeuVal Gly Pro Arg Asp Phe Val Ser Val Arg Cys Thr Lys Arg Arg 180 185 190Gly Ser Thr Cys Val Leu Ala Gly Met Ala Thr His Phe Gly Glu Met 195 200205 Pro Glu Gln Ser Gly Val Ile Arg Ala Glu His Gly Pro Thr Cys Met 210215 220 Val Leu His Pro Leu Ala Gly Ser Pro Ser Lys Thr Lys Leu Thr Trp225 230 235 240 Leu Leu Ser Ile Asp Leu Lys Gly Trp Leu Pro Lys Thr IleIle Asn 245 250 255 Gln Val Leu Ser Gln Thr Gln Ile Glu Phe Ala Asn HisLeu Arg Lys 260 265 270 Arg Leu Glu Ala 275 14 amino acids amino acidsingle linear peptide unknown 3 Ala Glu His Gly Pro Thr Cys Met Val LeuHis Pro Leu Ala 1 5 10 12 amino acids amino acid single linear peptideunknown 4 Ala Leu Gly Ile Leu Asn Asn Gln Glu Gly Trp Lys 1 5 10 19amino acids amino acid single linear peptide unknown 5 Gly Ser Thr CysVal Leu Ala Gly Met Ala Thr His Phe Gly Glu Met 1 5 10 15 Pro Glu Gln 6amino acids amino acid single linear peptide unknown 6 Asn Gln Glu GlyTrp Lys 1 5 9 amino acids amino acid single linear peptide unknown 7 AlaGlu His Gly Pro Thr Cys Met Val 1 5 11 amino acids amino acid singlelinear peptide unknown 8 Ile Leu Asn Asn Gln Glu Gly Trp Lys Lys Glu 1 510 25 base pairs nucleic acid single linear other nucleic acid /desc =“DNA” unknown modified_base one-of(3, 12, 15, 18) /mod_base= OTHER/note= “N = (A or C or G or T/U) or (unknown or other)” modified_base/mod_base= OTHER /note= “R = A or G” modified_base one-of(9, 21)/mod_base= OTHER /note= “Y = C or T/U” 9 GCNGARCAYG GNCCNACNTG YATGG 2525 base pairs nucleic acid single linear other nucleic acid /desc =“DNA” unknown modified_base one-of(5, 17) /mod_base= OTHER /note= “R = Aor G” modified_base one-of(8, 11, 14, 23) /mod_base= OTHER /note= “N =(A or C or G or T/U) or (unknown or other)” modified_base 20 /mod_base=OTHER /note= “Y = C or T/U” 10 CCATRCANGT NGGNCCRTGY TCNGC 25 17 basepairs nucleic acid single linear other nucleic acid /desc = “DNA”unknown modified_base /mod_base= OTHER /note= “Y = C or T/U”modified_base one-of(6, 9) /mod_base= OTHER /note= “R = A or G”modified_base 12 /mod_base= OTHER /note= “N = (A or C or G or T/U) or(unknown or other)” 11 AAYCARCARG GNTGGAA 17 17 base pairs nucleic acidsingle linear other nucleic acid /desc = “DNA” unknown 12 TTCCANCCYTCYTGRTT 17 401 base pairs nucleic acid single linear other nucleic acid/desc = “DNA” unknown 13 AACCAGGAAG GCTGGAAGAA GGAAAGCCAG CAGGAGAACGGGGACGAAGT GCTAAGTAAG 60 ATGGTGCCAG ATGTGGGCAA GGTGTTTCGC TTGGAGGTGGTGGTAGACCA GCCCATGGAC 120 AGACTCTATG AAGAACTTGT GGACCGCATG GAGGCCATGGGAGAGTGGAA CCCAAATGTC 180 AAGGAGATCA AGGTCCTGCA GAGGATTGGA AAAGACACGGTCATCACTCA TGAGCTGGCT 240 GCGGCGGCAG CAGGCAACCT GGTGGGGCCT CGAGACTTCGTGAGCGTGCG CTGTACCAAG 300 CGCAGAGGTT CCACCTGTGT GCTGGCAGGC ATGGCCACACATTTTGGGGA GATGCCGGAG 360 CAGAGTGGTG TCATCAGAGC TGAACACGGC CCCACCTGCA T401 1466 base pairs nucleic acid single linear other nucleic acid /desc= “RNA” unknown 14 GUCGACCCAC GCGUCCGCUC AGGACCUUGA AAGGCUCAGGAAGAACAACC CUUGAGCACC 60 UCAGCACUCA GCAUGUUCCU CGCUACGUUC AAGCUGUGUGCUGGAAGCUC CUAUAGACAU 120 AUGCGGAAUA UGAAAGGAUU AAGGCACCAA GCUGUGCUGGCCAUUGGCCA AGAGCUCAAC 180 UGGAGAGCAC UGGGGGAUUC CAGUCCCGGG UGGAUGGGUCAAGUUCGACG UCGGAGCUCU 240 CUGCUUGGUU CUCAACUGGA AGCAACACUC UAUAGUGACCAGGAGCUGUC CUACAUCCAG 300 CAGGGAGAGG UGGCUAUGCA GAAGGCCUUG GGCAUACUCAACAACCAGGA AGGCUGGAAG 360 AAGGAAAGCC AGCAGGAGAA CGGGGACGAA GUGCUAAGUAAGAUGGUGCC AGAUGUGGGC 420 AAGGUGUUUC GCUUGGAGGU GGUGGUAGAC CAGCCCAUGGACAGACUCUA UGAAGAACUU 480 GUGGACCGCA UGGAGGCCAU GGGAGAGUGG AACCCAAAUGUCAAGGAGAU CAAGGUCCUG 540 CAGAGGAUUG GAAAAGACAC GGUCAUCACU CAUGAGCUGGCUGCGGCGGC AGCAGGCAAC 600 CUGGUGGGGC CUCGAGACUU CGUGAGCGUG CGCUGUACCAAGCGCAGAGG UUCCACCUGU 660 GUGCUGGCAG GCAUGGCCAC ACAUUUUGGG GAGAUGCCGGAGCAGAGUGG UGUCAUCAGA 720 GCUGAACACG GCCCCACCUG CAUGGUGCUU CAUCCACUGGCUGGAAGUCC CUCCAAGACU 780 AAACUCACUU GGCUGCUCAG UAUUGACCUG AAGGGGUGGCUGCCGAAGAC AAUCAUCAAC 840 CAGGUCCUAU CGCAGACCCA GAUAGAGUUC GCCAACCACCUGCGCAAGCG CCUGGAAGCC 900 AGCCCUGCCU CUGAGGCCCA GUGUUAAGGA CUGUCCACCACAUUGACCUG CAAAUCAUUG 960 GAAGCUCUCA CAGGAAGCCU GCAAGUCUGU CCAUCUUCAGCUAACAGCAU CGGGAGGGGU 1020 GGUAGUCAGG AGACACUAGG ACUGACUGGU AAAAUCAGGAUCAGCAAAAU AGAAAUGAGG 1080 CUUAGAAUAA AAGUUCUCUA GUGUCUCCCA CUGCAUAGCUGUGAAGGCUA AGGGAUAAGU 1140 AGCUAUGAAA CCUUUCAUCU AGGCUUGUAU AUGCUGACCUAAAAGACACC AGCAGCUACG 1200 AACAGGGGAU GCUAAGGAUC GGGAACUGUU GUCUUACCAGCUCCAAAUGU CACUACCUGA 1260 AGGCAGUGUG CACACAAAGC AAGGUCUUGC CUAGGAAACUCUGUAAAAGU UCUCCUCUGU 1320 AAAAGGCCAG AACUUGAAUG AAACUACCUA CAAAGGGCCUUUCCAGAGUA UUCCAACUUU 1380 UCUCUGAGGA GAAAUGAAAC CAUCAUUGUG CCGACUUCCCUACUAAUCCC AUGACAAUAA 1440 AGAACAUACA UAAAAAAAAA AAAAAA 1466 722 basepairs nucleic acid single linear unknown 15 GTGGATTAAC CAGGTTCGGCGGCGGAGCTC TCTACTCGGT TCTCGGCTGG AAGAGACTCT 60 CTACAGTGAC CAGGAGCTGGCCTATCTCCA GCAGGGGGAG GAGGCCATGC AGAAGGCCTT 120 GGGCATCCTT AGCAACCAAGAGGGCTGGAA GAAGGAGAGT CAGCAGGACA ATGGGGACAA 180 AGTGATGAGT AAAGTGGTCCCAGATGTGGG CAAGGTGTTC CGGCTGGAGG TCGTGGTGGA 240 CCAGCCCATG GAGAGGCTCTATGAAGAGCT CGTGGAGCGC ATGGAAGCAA TGGGGGAGTG 300 GAACCCCAAT GTCAAGGAGATCAAGGTCCT GCAGAAGATC GGAAAAGATA CATTCATTAC 360 TCACGAGCTG GCTGCCGAGGCAGCAGGAAA CCTGGTGGGG CCCCGTGACT TTGTGAGCGT 420 GCGCTGTGCC AAGCGCCGAGGCTCCACCTG TGTGCTGGCT GGCATGGACA CAGACTTCGG 480 GAACATGCCT GAGCAGAAGGGTGTCATCAG GGCGGAGCAC GGTCCCACTT GCATGGTGCT 540 TCACCCGTTG GCTGGAAGTCCCTCTAAGAC CAAACTTACG TGGCTACTCA GCATCGACCT 600 CAAGGGGTGG CTGCCCAAGAGCATCATCAA CCAGGTCCTG TCCCAGACCC AGGTGGATTT 660 TGCCAACCAC CTGCGCAAGCGCCTGGAGTC CCACCCTGCC TCTGAAGCCA GGTGTTGAAG 720 AC 722 134 base pairsnucleic acid single linear unknown 16 ATGCTGCTAG CGACATTCAA GCTGTGCGCTGGGAGCTCCT ACAGACACAT GCGCAACATG 60 AAGGGGCTGA GGCAACAGGC TGTGATGGCCATCAGCCAGG AGCTGAACCG GAGGGCCCTG 120 GGGGGCCCCA CCCC 134 19 base pairsnucleic acid single linear unknown 17 ACTGGAAGCC TGCAAGTCT 19 285 aminoacids amino acid linear unknown 18 Met Leu Leu Ala Thr Phe Lys Leu CysAla Gly Ser Ser Tyr Arg His 1 5 10 15 Met Arg Asn Met Lys Gly Leu ArgGln Gln Ala Val Met Ala Ile Ser 20 25 30 Gln Glu Leu Asn Arg Arg Ala LeuGly Gly Pro Thr Pro Ser Thr Trp 35 40 45 Ile Asn Gln Val Arg Arg Arg SerSer Leu Leu Gly Ser Arg Leu Glu 50 55 60 Glu Thr Leu Tyr Ser Asp Gln GluLeu Ala Tyr Leu Gln Gln Gly Glu 65 70 75 80 Glu Ala Met Gln Lys Ala LeuGly Ile Leu Ser Asn Gln Glu Gly Trp 85 90 95 Lys Lys Glu Ser Gln Gln AspAsn Gly Asp Lys Val Met Ser Lys Val 100 105 110 Val Pro Asp Val Gly LysVal Phe Arg Leu Glu Val Val Val Asp Gln 115 120 125 Pro Met Glu Arg LeuTyr Glu Glu Leu Val Glu Arg Met Glu Ala Met 130 135 140 Gly Glu Trp AsnPro Asn Val Lys Glu Ile Lys Val Leu Gln Lys Ile 145 150 155 160 Gly LysAsp Thr Phe Ile Thr His Glu Leu Ala Ala Glu Ala Ala Gly 165 170 175 AsnLeu Val Gly Pro Arg Asp Phe Val Ser Val Arg Cys Ala Lys Arg 180 185 190Arg Gly Ser Thr Cys Val Leu Ala Gly Met Ala Thr Asp Phe Gly Asn 195 200205 Met Pro Glu Gln Lys Gly Val Ile Arg Ala Glu His Gly Pro Thr Cys 210215 220 Met Val Leu His Pro Leu Ala Gly Ser Pro Ser Lys Thr Lys Leu Thr225 230 235 240 Trp Leu Leu Ser Ile Asp Leu Lys Gly Trp Leu Pro Lys SerIle Ile 245 250 255 Asn Gln Val Leu Ser Gln Thr Gln Val Asp Phe Ala AsnHis Leu Arg 260 265 270 Lys Arg Leu Glu Ser His Pro Ala Ser Glu Ala ArgCys 275 280 285 1641 base pairs nucleic acid single linear unknown 19AGAACACCAG GTCCAGGCTG CAGCTGCGGG ACTCAGAGGC GAACGTTGAG GGGCTCAGGA 60AGGACGAAGA ACCACCCTTG AGAGAAGAGG CAGCAGCAGC GCGGCAGCAG CAGCGGCAGC 120GACCCCACCA CTGCCACATT TGCCAGGAAA CAATGCTGCT AGCGACATTC AAGCTGTGCG 180CTGGGAGCTC CTACAGACAC ATGCGCAACA TGAAGGGGCT GAGGCAACAG GCTGTGATGG 240CCATCAGCCA GGAGCTGAAC CGGAGGGCCC TGGGGGGCCC CACCCCTAGC ACGTGGATTA 300ACCAGGTTCG GCGGCGGAGC TCTCTACTCG GTTCTCGGCT GGAAGAGACT CTCTACAGTG 360ACCAGGAGCT GGCCTATCTC CAGCAGGGGG AGGAGGCCAT GCAGAAGGCC TTGGGCATCC 420TTAGCAACCA AGAGGGCTGG AAGAAGGAGA GTCAGCAGGA CAATGGGGAC AAAGTGATGA 480GTAAAGTGGT CCCAGATGTG GGCAAGGTGT TCCGGCTGGA GGTCGTGGTG GACCAGCCCA 540TGGAGAGGCT CTATGAAGAG CTCGTGGAGC GCATGGAAGC AATGGGGGAG TGGAACCCCA 600ATGTCAAGGA GATCAAGGTC CTGCAGAAGA TCGGAAAAGA TACATTCATT ACTCACGAGC 660TGGCTGCCGA GGCAGCAGGA AACCTGGTGG GGCCCCGTGA CTTTGTGAGC GTGCGCTGTG 720CCAAGCGCCG AGGCTCCACC TGTGTGCTGG CTGGCATGGC CACAGACTTC GGGAACATGC 780CTGAGCAGAA GGGTGTCATC AGGGCGGAGC ACGGTCCCAC TTGCATGGTG CTTCACCCGT 840TGGCTGGAAG TCCCTCTAAG ACCAAACTTA CGTGGCTACT CAGCATCGAC CTCAAGGGGT 900GGCTGCCCAA GAGCATCATC AACCAGGTCC TGTCCCAGAC CCAGGTGGAT TTTGCCAACC 960ACCTGCGCAA GCGCCTGGAG TCCCACCCTG CCTCTGAAGC CAGGTGTTGA AGACCAGCCT 1020GCTGTTCCCA ACTGTGCCCA GCTGCACTGG TACACACGCT CATCAGGAGA ATCCCTACTG 1080GAAGCCTGCA AGTCTAAGAT CTCCATCTGG TGACAGTGGG ATGGGTGGGG TTCGTGTTTA 1140GAGTATGACA CTAGGATTCA GATTGGTGAA AGTTTTTAGT ACCAAGAAAA CAGGGATGAG 1200CTCTTGGATT AAAAGGTAAC TTCATTCACT GATTAGCTAT GACATGAGGG TTCAGGCCCG 1260CTAAAAATAA TTGTAAAACT TTTTTTCTGG GCCCTTATGT ACCCACCTAA AACCATCTTT 1320AAAATGCTAG TGGCTGATAT GGGTGTGGGG GATGCTAACC ACAGGGCCTG AGAAGTCTTG 1380CTTTATGGGC TCAAGAATGC CATGCGCTGG CAGTACATGT GCACAAAGCA GAATCTCAGA 1440GGGTCTCCTG CAGCCCTCTG CTCCTCCCGG CCGCTGCACA GCAACACCAC AGAACAAGCA 1500GCACCCCACA GTGGGTGCCT TCCAGAAATA TAGTCCAAGC TTTCTCTGTG GAAAAAGACA 1560AAACTCATTA GTAGACATGT TTCCCTATTG CTTTCATAGG CACCAGTCAG AATAAAGAAT 1620CATAATTCAC ACAAAAAAAA A 1641

What is claimed is:
 1. A purified nucleic acid molecule having anucleotide sequence comprising 70% to 90% of nucleotides consisting ofthe sequence SEQ ID NO: 14.